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Sample records for human deoxycytidine kinase

  1. Modulation of human UMP/CMP kinase affects activation and cellular sensitivity of deoxycytidine analogs.

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    Liou, Jieh-Yuan; Lai, Hui-Ru; Hsu, Chih-Hung; Chang, Wei-Ling; Hsieh, Mei-Ju; Huang, Yu-Chun; Cheng, Yung-Chi

    2010-02-01

    Deoxycytidine analogs are an important class of clinically active antiviral and anticancer agents. The stepwise phosphorylation of these analogs to triphosphate metabolites is crucial for biological action. Human UMP/CMP kinase (UMP/CMPK; cytidylate kinase; EC 2.7.4.14) is thought to be responsible for phosphorylation of UMP, CMP, and dCMP and may also play an important role in the activation of pyrimidine analogs. However, no evidence has verified this notion in intact cells. In this study we explored the functional roles of UMP/CMPK in natural pyrimidine synthesis and metabolism of deoxycytidine analogs, as well as 5-FU in HeLa S3 and HCT8 cells. The amounts of UMP/CMPK protein in different cell lines correlated with UMP, CMP, and dCMP kinase activities and amounts of UMP/CMPK RNA. Modulation of UMP/CMPK by overexpression or down-regulation had no impact on natural pyrimidine nucleotides and cell growth. However, down-regulating UMP/CMPK expression by siRNA led to a decrease in the formation of the triphosphate metabolites, resulting in cellular resistance to these analogs. More diphosphate and triphosphate metabolites of deoxycytidine analogs were detected and cellular sensitivity to these agents was increased in the UMP/CMPK-overexpressing cells. This study indicates that the second step enzyme (UMP/CMPK) is responsible for the phosphorylation of pyrimidine analogs and also has an impact on cellular sensitivity to these analogs in those cell lines.

  2. Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

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    Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan; Konrad, Manfred; Lavie, Arnon; (UIC); (MXPL-G)

    2009-03-04

    Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-type dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.

  3. Identification of phosphorylation sites on human deoxycytidine kinase after overexpression in eucaryotic cells.

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    Smal, C; Vertommen, D; Bertrand, L; Rider, M H; van den Neste, E; Bontemps, F

    2006-01-01

    Compelling evidence suggests that deoxycytidine kinase (dCK), a key enzyme in the salvage of deoxyribonucleosides and in the activation of clinically relevant nucleoside analogues, can be regulated by reversible phosphorylation. In this study, we show that dCK overexpressed in HEK-293T cells was labelled after incubation of the cells with [32P]orthophosphate. Tandem mass spectrometry allowed the identification of 4 in vivo phosphorylation sites, Thr3, Ser11, Ser15, and Ser74. These results provide the first evidence that dCK is constitutively multiphosphorylated in intact cells. In addition, site-directed mutagenesis demonstrated that phosphorylation of Ser74, the major in vivo phosphorylation site, is crucial for dCK activity.

  4. Potent Sensitisation of Cancer Cells to Anticancer Drugs by a Quadruple Mutant of the Human Deoxycytidine Kinase.

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    Safiatou T Coulibaly

    Full Text Available Identifying enzymes that, once introduced in cancer cells, lead to an increased efficiency of treatment constitutes an important goal for biomedical applications. Using an original procedure whereby mutant genes are generated based on the use of conditional lentivector genome mobilisation, we recently described, for the first time, the identification of a human deoxycytidine kinase (dCK mutant (G12 that sensitises a panel of cancer cell lines to treatment with the dCK analogue gemcitabine. Here, starting from the G12 variant itself, we generated a new library and identified a mutant (M36 that triggers even greater sensitisation to gemcitabine than G12. With respect to G12, M36 presents an additional mutation located in the region that constitutes the interface of the dCK dimer. The simple presence of this mutation halves both the IC50 and the proportion of residual cells resistant to the treatment. Furthermore, the use of vectors with self-inactivating LTRs leads to an increased sensitivity to treatment, a result compatible with a relief of the transcriptional interference exerted by the U3 promoter on the internal promoter that drives the expression of M36. Importantly, a remarkable effect is also observed in treatments with the anticancer compound cytarabine (AraC, for which a 10,000 fold decrease in IC50 occurred. By triggering the sensitisation of various cancer cell types with poor prognosis to two commonly used anticancer compounds M36 is a promising candidate for suicide gene approaches.

  5. The relation between deoxycytidine kinase activity and the radiosensitising effect of gemcitabine in eight different human tumour cell lines

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    Peters Godefridus J

    2006-05-01

    Full Text Available Abstract Background Gemcitabine (dFdC is an active antitumour agent with radiosensitising properties, shown both in preclinical and clinical studies. In the present study, the relation between deoxycytidine kinase (dCK activity and the radiosensitising effect of gemcitabine was investigated in eight different human tumour cell lines. Methods Tumour cells were treated with dFdC (0–100 nM for 24 h prior to radiotherapy (RT (γ-Co60, 0–6 Gy, room temperature. Cell survival was determined 7, 8, or 9 days after RT by the sulforhodamine B test. dCK activity of the cells was determined by an enzyme activity assay. Results A clear concentration-dependent radiosensitising effect of dFdC was observed in all cell lines. The degree of radiosensitisation was also cell line dependent and seemed to correlate with the sensitivity of the cell line to the cytotoxic effect of dFdC. The dCK activity of our cell lines varied considerably and differed up to three fold from 5 to 15 pmol/h/mg protein between the tested cell lines. In this range dCK activity was only weakly related to radiosensitisation (correlation coefficient 0.62, p = 0.11. Conclusion Gemcitabine needs to be metabolised to the active nucleotide in order to radiosensitise the cells. Since dFdCTP accumulation and incorporation into DNA are concentration dependent, the degree of radiosensitisation seems to be related to the extent of dFdCTP incorporated into DNA required to inhibit DNA repair. The activity of dCK does not seem to be the most important factor, but is clearly a major factor. Other partners of the intracellular metabolism of gemcitabine in relation to the cell cycle effects and DNA repair could be more responsible for the radiosensitising effect than dCK activity.

  6. Casein kinase 1delta activates human recombinant deoxycytidine kinase by Ser-74 phosphorylation, but is not involved in the in vivo regulation of its activity.

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    Smal, Caroline; Vertommen, Didier; Amsailale, Rachid; Arts, Angélique; Degand, Hervé; Morsomme, Pierre; Rider, Mark H; Neste, Eric Van Den; Bontemps, Françoise

    2010-10-01

    Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI delta. We showed that recombinant CKI delta phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI delta correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI delta, strengthening the key role of this residue in the control of dCK activity. However, neither CKI delta inhibitors nor CKI delta siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI delta in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI delta could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme. Copyright 2010 Elsevier Inc. All rights reserved.

  7. Expression Levels of Human Equilibrative Nucleoside Transporter 1 and Deoxycytidine Kinase Enzyme as Prognostic Factors in Patients with Acute Myeloid Leukemia Treated with Cytarabine.

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    Candelaria, Myrna; Corrales-Alfaro, Carmen; Gutiérrez-Hernández, Olga; Díaz-Chavez, José; Labardini-Méndez, Juan; Vidal-Millán, Silvia; Herrera, Luis A

    2016-01-01

    Cytarabine (Ara-C) is the primary drug in different treatment schemas for acute myeloid leukemia (AML) and requires the human equilibrative nucleoside transporter (hENT1) to enter cells. The deoxycytidine kinase (dCK) enzyme limits its activation rate. Therefore, decreased expression levels of these genes may influence the response rate to this drug. AML patients without previous treatment were enrolled. The expression of hENT1 and dCK genes was analyzed using RT-PCR. Clinical parameters were registered. All patients received Ara-C + doxorubicin as an induction regimen (7 + 3 schema). Descriptive statistics were used to analyze data. Uni- and multivariate analyses were performed to determine factors that influenced response and survival. Twenty-eight patients were included from January 2011 until December 2012. Median age was 36.5 years. All patients had an adequate performance status (43% with ECOG 1 and 57% with ECOG 2). Cytogenetic risk was considered unfavorable in 54% of the patients. Complete response was achieved in 53.8%. Cox regression analysis showed that a higher hENT1 expression level was the only factor that influenced response and survival. These results highly suggest that the pharmacogenetic analyses of Ara-C influx may be decisive in AML patients. © 2016 S. Karger AG, Basel.

  8. [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

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    Kim, Woosuk; Le, Thuc M; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T; Abt, Evan R; Capri, Joseph R; Austin, Wayne R; Van Valkenburgh, Juno S; Steele, Dalton; Gipson, Raymond M; Slavik, Roger; Cabebe, Anthony E; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S; Lee, Jason T; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F; Witte, Owen N; Donahue, Timothy R; Phelps, Michael E; Herschman, Harvey R; Herrmann, Ken; Czernin, Johannes; Radu, Caius G

    2016-04-12

    Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

  9. [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity

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    Kim, Woosuk; Le, Thuc M.; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T.; Abt, Evan R.; Capri, Joseph R.; Austin, Wayne R.; Van Valkenburgh, Juno S.; Steele, Dalton; Gipson, Raymond M.; Slavik, Roger; Cabebe, Anthony E.; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S.; Lee, Jason T.; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F.; Witte, Owen N.; Donahue, Timothy R.; Phelps, Michael E.; Herschman, Harvey R.; Herrmann, Ken; Czernin, Johannes; Radu, Caius G.

    2016-01-01

    Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds—[18F]Clofarabine; 2-chloro-2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-adenine ([18F]CFA) and 2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-guanine ([18F]F-AraG)—for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [18F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [18F]F-AraG is a better substrate for dGK than for dCK. [18F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [18F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [18F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [18F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [18F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [18F]CFA PET as a new cancer biomarker for treatment stratification and monitoring. PMID:27035974

  10. Staurosporine increases toxicity of gemcitabine in non-small cell lung cancer cells: role of protein kinase C, deoxycytidine kinase and ribonucleotide reductase

    NARCIS (Netherlands)

    Sigmond, Jennifer; Bergman, Andries M.; Leon, Leticia G.; Loves, Willem J. P.; Hoebe, Eveline K.; Peters, Godefridus J.

    2010-01-01

    Gemcitabine, a deoxycytidine analog, active against non-small cell lung cancer, is phosphorylated by deoxycytidine kinase (dCK) to active nucleotides. Earlier, we found increased sensitivity to gemcitabine in P-glycoprotein (SW-2R160) and multidrug resistance-associated protein (SW-2R120),

  11. Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser-74 dephosphorylation.

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    Amsailale, Rachid; Beyaert, Maxime; Smal, Caroline; Janssens, Veerle; Van Den Neste, Eric; Bontemps, Françoise

    2014-03-03

    Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn(2+)-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Hypoxia-induced deoxycytidine kinase contributes to epithelial proliferation in pulmonary fibrosis.

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    Weng, Tingting; Poth, Jens M; Karmouty-Quintana, Harry; Garcia-Morales, Luis J; Melicoff, Ernestina; Luo, Fayong; Chen, Ning-yuan; Evans, Christopher M; Bunge, Raquel R; Bruckner, Brian A; Loebe, Matthias; Volcik, Kelly A; Eltzschig, Holger K; Blackburn, Michael R

    2014-12-15

    Idiopathic pulmonary fibrosis (IPF) is a deadly lung disease with few therapeutic options. Apoptosis of alveolar epithelial cells, followed by abnormal tissue repair characterized by hyperplastic epithelial cell formation, is a pathogenic process that contributes to the progression of pulmonary fibrosis. However, the signaling pathways responsible for increased proliferation of epithelial cells remain poorly understood. To investigate the role of deoxycytidine kinase (DCK), an important enzyme for the salvage of deoxynucleotides, in the progression of pulmonary fibrosis. DCK expression was examined in the lungs of patients with IPF and mice exposed to bleomycin. The regulation of DCK expression by hypoxia was studied in vitro and the importance of DCK in experimental pulmonary fibrosis was examined using a DCK inhibitor and alveolar epithelial cell-specific knockout mice. DCK was elevated in hyperplastic alveolar epithelial cells of patients with IPF and in mice exposed to bleomycin. Increased DCK was localized to cells associated with hypoxia, and hypoxia directly induced DCK in alveolar epithelial cells in vitro. Hypoxia-induced DCK expression was abolished by silencing hypoxia-inducible factor 1α and treatment of bleomycin-exposed mice with a DCK inhibitor attenuated pulmonary fibrosis in association with decreased epithelial cell proliferation. Furthermore, DCK expression, and proliferation of epithelial cells and pulmonary fibrosis was attenuated in mice with conditional deletion of hypoxia-inducible factor 1α in the alveolar epithelium. Our findings suggest that the induction of DCK after hypoxia plays a role in the progression of pulmonary fibrosis by contributing to alveolar epithelial cell proliferation.

  13. Bystander killing of malignant cells via the delivery of engineered thymidine-active deoxycytidine kinase for suicide gene therapy of cancer.

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    Neschadim, A; Wang, J C M; Lavie, A; Medin, J A

    2012-05-01

    Activity and specificity of chemotherapeutic agents against solid tumors can be augmented via the targeted or localized delivery of 'suicide' genes. Selective activation of specific prodrugs in cells expressing the 'suicide' gene drives their elimination by apoptosis, while also enabling the killing of adjacent bystander cells. Strong bystander effects can compensate for poor 'suicide' gene delivery, and depend on the prodrugs used and mechanisms for the acquisition of activated drug by the bystander population, such as the presence of gap junctional intercellular communications. Although a number of 'suicide' gene therapies for cancer have been developed and characterized, such as herpes simplex virus-derived thymidine kinase (HSV-tk)-based activation of ganciclovir, their limited success highlights the need for the development of more robust approaches. Limiting activation kinetics and evolution of chemoresistance are major obstacles. Here we describe 'suicide' gene therapy of cancer based on the lentivirus-mediated delivery of a thymidine-active human deoxycytidine kinase variant. This enzyme possesses substrate plasticity that enables it to activate a multitude of prodrugs, some with distinct mechanisms of action. We evaluated the magnitude and mechanisms of bystander effects induced by different prodrugs, and show that when used in combination, they can synergistically enhance the bystander effect while avoiding off-target toxicity.

  14. Aberrant Gene Expression in Human Non Small Cell Lung Carcinoma Cells Exposed to Demethylating Agent 5-Aza-2'-Deoxycytidine

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    Bao-Zhu Yuan

    2004-07-01

    Full Text Available The identification of genes undergoing genetic or epigenetic alterations and contributing to the development of cancer is critical to our understanding of the molecular mechanisms of carcinogenesis. A new approach in identifying alterations of genes that might be relevant to the process of tumor development was used in this study by examining the gene expression profile in human lung cancer cells exposed to 5-aza-2'deoxycytidine (5-aza-dC. A cDNA array analysis was carried out on 5-aza-dC-treated and untreated non small cell lung cancer (NSCLC cell line NCI-H522. Sixteen and 14 genes were upregulated and downregulated, respectively, by 5-aza-dC treatment. Among them, downregulation of tyrosine protein kinase ABL2 (ABL2 gene and upregulation of hint/protein kinase C inhibitor 1 (Hint/PKCI-1, DVL1, TIMP-1, and TRP-1 genes were found in expanded observations in two or three of five 5-aza-dC-treated NSCLC cell lines. Among these genes, we found that cDNA transfer of Hint/PKCI-1 resulted in a significant in vitro growth inhibition in two cell lines exhibiting 5-aza-dC-induced upregulation of Hint/PKCI-1 and significantly reduced in vivo tumorigenicity of one NSCLC cell line. Hint/PKCI-1, which is the only other characterized human histidine triad (HIT nucleotide-binding protein in addition to tumor-suppressor gene FHIT, might be involved in lung carcinogenesis.

  15. Paclitaxel alters the expression and specific activity of deoxycytidine kinase and cytidine deaminase in non-small cell lung cancer cell lines

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    Patel Shitalben R

    2009-06-01

    Full Text Available Abstract Background We observed that paclitaxel altered the pharmacokinetic properties of gemcitabine in patients with non-small cell lung cancer (NSCLC and limited the accumulation of gemcitabine and its metabolites in various primary and immortalized human cells. Therefore, we classified the drug-drug interaction and the effects of paclitaxel on deoxycytidine kinase (dCK and cytidine deaminase (CDA in three NSCLC cell lines. These enzymes are responsible for the metabolism of gemcitabine to its deaminated metabolite dFdU (80% of the parent drug and the phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP. These metabolites appear to relate to sensitivity and tolerability of gemcitabine based on previous animal and laboratory studies. Methods Three immortalized human cells representative of the most common histological subtypes identified in patients with advanced NSCLC were exposed to the individual drugs or combinations to complete a multiple drug effect analysis. These same cell lines were exposed to vehicle-control or paclitaxel and the mRNA levels, protein expression and specific activity of dCK and CDA were compared. Comparisons were made using a two-tailed paired t-test or analysis of variance with a P value of Results The multiple drug effect analysis indicated synergy for H460, H520 and H838 cells independent of sequence. As anticipated, paclitaxel-gemcitabine increased the number of G2/M cells, whereas gemcitabine-paclitaxel increased the number of G0/G1 or S cells. Paclitaxel significantly decreased dCK and CDA mRNA levels in H460 and H520 cells (40% to 60%, P Conclusion In summary, paclitaxel altered the mRNA levels and specific activity of dCK and CDA and these effects could be dependent on histological subtype. More cell and animal studies are needed to further characterize the relationship between mRNA levels and the overall drug-drug interaction and the potential to use histological subtype as a predictive factor in the

  16. Impact of 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate for induction of human regulatory T cells.

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    Kehrmann, Jan; Tatura, Roman; Zeschnigk, Michael; Probst-Kepper, Michael; Geffers, Robert; Steinmann, Joerg; Buer, Jan

    2014-07-01

    The epigenetic regulation of transcription factor genes is critical for T-cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3 and suppressive function. We analysed the impact of hypomethylating agents 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate on human CD4(+)  CD25(-) T cells for generating demethylation within FOXP3-TSDR and inducing functional Treg cells. Gene expression, including lineage-specifying transcription factors of the major T-cell lineages and their leading cytokines, functional properties and global transcriptome changes were analysed. The FOXP3-TSDR methylation pattern was determined by using deep amplicon bisulphite sequencing. 5-aza-2'-deoxycytidine induced FOXP3-TSDR hypomethylation and expression of the Treg-cell-specific genes FOXP3 and LRRC32. Proliferation of 5-aza-2'-deoxycytidine-treated cells was reduced, but the cells did not show suppressive function. Hypomethylation was not restricted to FOXP3-TSDR and expression of master transcription factors and leading cytokines of T helper type 1 and type 17 cells were induced. Epigallocatechin-3-gallate induced global DNA hypomethylation to a lesser extent than 5-aza-2'-deoxycitidine, but no relevant hypomethylation within FOXP3-TSDR or expression of Treg-cell-specific genes. Neither of the DNA methyltransferase inhibitors induced fully functional human Treg cells. 5-aza-2'-deoxycitidine-treated cells resembled Treg cells, but they did not suppress proliferation of responder cells, which is an essential capability to be used for Treg cell transfer therapy. Using a recently developed targeted demethylation technology might be a more promising approach for the generation of functional Treg cells. © 2014 John Wiley & Sons Ltd.

  17. Delineating the effect of demethylating agent 5-aza-2'-deoxycytidine on human Caco-2 colonic carcinoma cells.

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    Li, Xiumei; Qin, Bingzhao; Liu, B O

    2016-07-01

    Aberrant epigenetic changes are known to contribute to various phases of tumor development. The gene function loss caused by aberrant methylation is analogous to genetic mutations. Unlike genetic mutations, epigenetic alterations can be reversed. 5-Aza-2'-deoxycytidine (5-aza-CdR) has been approved by the Food and Drug Administration for the treatment of certain types of cancer, such as MDS and leukemia. The aim of the present study was to determine whether 5-aza-CdR has the potential to be used in the treatment of colon cancer using a human Caco-2 colonic carcinoma cell line. The effect of 5-aza-CdR on cell proliferation, cell cycle, apoptosis and reversal of aberrant methylation of the Ras association domain family 1A (RASSF1A) gene was also examined. The 5-aza-CdR was prepared at different concentrations in sterile tri-distilled water at 0.4, 1.6, 6.4, 25.6 and 102.4 µmol/l and employed to treat the human Caco-2 colonic carcinoma cells. An MTT assay was used to detect the effect of 5-aza-CdR on cell proliferation. Flow cytometry was used to examine the cell cycle and apoptosis. The RASSF1A mRNA transcript level was examined by reverse transcription-polymerase chain reaction. The results showed that 5-aza-CdR inhibited the proliferation of Caco-2 cells in a time- and concentration-dependent manner (pCaco-2 cells. In conclusion, 5-aza-CdR inhibited growth and promoted apoptosis in Caco-2 cells by upregulating the epigenetically silenced tumor suppressor RASSF1A gene.

  18. Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1

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    Skovgaard, Tine; Uhlin, Ulla; Munch-Petersen, Birgitte

    2012-01-01

    The first step for the intracellular retention of several anticancer or antiviral nucleoside analogues is the addition of a phosphate group catalysed by a deoxyribonucleoside kinase such as thymidine kinase 1 (TK1). Recently, human TK1 (HuTK1) has been crystallized and characterized using different...... ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket...... surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3...

  19. The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

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    J. Zych

    2013-05-01

    Full Text Available Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs derived from bone marrow (BM-MSCs and adipose tissue (ADSCs using the chromatin-modifying agents trichostatin A (TSA, a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC, a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

  20. A mathematical model of human thymidine kinase 2 activity

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    Radivoyevitch, Tom; Munch-Petersen, Birgitte; Wang, Liya

    2011-01-01

    _ The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ∼0.5 (apparent negative cooperati......_ The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ∼0.5 (apparent negative...

  1. GSTP1 DNA methylation and expression status is indicative of 5-aza-2'-deoxycytidine efficacy in human prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Karen Chiam

    Full Text Available DNA methylation plays an important role in carcinogenesis and the reversibility of this epigenetic modification makes it a potential therapeutic target. To date, DNA methyltransferase inhibitors (DNMTi have not demonstrated clinical efficacy in prostate cancer, with one of the major obstacles being the inability to monitor drug activity during the trial. Given the high frequency and specificity of GSTP1 DNA methylation in prostate cancer, we investigated whether GSTP1 is a useful marker of DNMTi treatment efficacy. LNCaP prostate cancer cells were treated with 5-aza-2'-deoxycytidine (5-aza-CdR either with a single high dose (5-20 µM, every alternate day (0.1-10 µM or daily (0.005-2.5 µM. A daily treatment regimen with 5-aza-CdR was optimal, with significant suppression of cell proliferation achieved with doses of 0.05 µM or greater (p<0.0001 and induction of cell death from 0.5 µM (p<0.0001. In contrast, treatment with a single high dose of 20 µM 5-aza-CdR inhibited cell proliferation but was not able to induce cell death. Demethylation of GSTP1 was observed with doses of 5-aza-CdR that induced significant suppression of cell proliferation (≥ 0.05 µM. Re-expression of the GSTP1 protein was observed only at doses of 5-aza-CdR (≥ 0.5 µM associated with induction of cell death. Treatment of LNCaP cells with a more stable DNMTi, Zebularine required at least a 100-fold higher dose (≥ 50 µM to inhibit proliferation and was less potent in inducing cell death, which corresponded to a lack of GSTP1 protein re-expression. We have shown that GSTP1 DNA methylation and protein expression status is correlated with DNMTi treatment response in prostate cancer cells. Since GSTP1 is methylated in nearly all prostate cancers, our results warrant its testing as a marker of epigenetic therapy response in future clinical trials. We conclude that the DNA methylation and protein expression status of GSTP1 are good indicators of DNMTi efficacy.

  2. Human Leukocyte Antigen-G Is Frequently Expressed in a Multicentric Study on Glioblastoma and May Be Induced in Vitro by Combined 5-aza-2'-deoxycytidine and Interferon-γ Treatments

    DEFF Research Database (Denmark)

    Wastowski, Isabela J; Simões, Renata T; Yaghi, Layale

    2012-01-01

    Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex (MHC) class I molecule involved in immune tolerance processes, playing an important role in the maintenance of the semi-allogeneic fetus. Although HLA-G expression is restricted in normal tissues, it is broadly......-G protein expression was associated with a better long-term survival rate. The mechanisms underlying HLA-G gene expression were investigated in glioma cell lines U251MG, D247MG, and U138MG. Induction of HLA-G transcriptional activity was dependent of 5-aza-2'-deoxycytidine treatment and enhanced...... by interferon-γ. HLA-G protein expression was observed in U251MG cells only. These cells exhibited a permissive chromatin state at the HLA-G gene promoter and the highest levels of induced HLA-G transcriptional activity following 5-aza-2'-deoxycytidine treatment. Several antigen-presenting machinery components...

  3. Combined Treatment with Valproic Acid and 5-Aza-2'-Deoxycytidine Synergistically Inhibits Human Clear Cell Renal Cell Carcinoma Growth and Migration.

    Science.gov (United States)

    Xi, Wenjin; Chen, Xu; Sun, Jinbo; Wang, Wei; Huo, Yi; Zheng, Guoxu; Wu, Jieheng; Li, Yufang; Yang, Angang; Wang, Tao

    2018-02-19

    BACKGROUND Histone acetylation and DNA methylation are important mammalian epigenetic modifications that participate in the regulation of gene expression. Because dysregulation of histone deacetylase and DNA methyltransferases are hallmarks of malignancy, they have become promising therapeutic targets. In this study, we explored the anti-tumor activity of valproic acid (VPA), a histone deacetylase inhibitor (HDACi) and 5-Aza-2'-deoxycytidine (5-Aza), an inhibitor of DNA methyltransferases, on renal cell carcinoma (RCC) cell lines 786-O and 769-P. MATERIAL AND METHODS The cell proliferation was detected by xCELLigence RTCA DP Instrument, viability by CCK8 assay, cell apoptosis and cell cycle by flow cytometry, and cell migration by wound healing assay, Transwell assay and xCELLigence RTCA DP Instrument. RESULTS We discovered that VPA and 5-Aza could individually induce decreased viability and have an inhibitory effect on the proliferation of 786-O and 769-P cells. This anti-growth effect was more pronounced when the cells were treated with both VPA and 5-Aza. The combination of VPA and 5-Aza also elicited more apoptosis and produced more cell cycle arrest in the G1 phase for both cell lines. On the other hand, treatment of RCC cells with VPA, 5-Aza, or a combination of both resulted in slow wound healing and impaired migration. CONCLUSIONS These findings clearly demonstrated that VPA combined with 5-Aza could significantly increase anti-RCC effects by inhibiting cellular proliferation, inducing apoptosis, promoting cell cycle arrest and prohibiting the migration of human RCC cells.

  4. Synergistic antineoplastic action of 5-aza-2'deoxycytidine (decitabine in combination with different inhibitors of enhancer of zeste homolog 2 (EZH2 on human lung carcinoma cells

    Directory of Open Access Journals (Sweden)

    Nascimento ASF

    2016-10-01

    Full Text Available Patients with metastatic lung cancer have a very poor prognosis indicating an urgent need to develop more effective chemotherapy. Aberrant promoter DNA methylation can result in the epigenetic silencing of tumor suppressor genes (TSGs in lung cancer. 5-Aza-2’deoxycytidine (5-Aza-CdR, decitabine, an inhibitor of DNA methylation, is able to reactivate silent TSGs. Trimethylation of histone H3 on lysine 27 (H3K27me3 by enhancer of zeste homolog 2 (EZH2 histone methyltransferase can also silence TSGs in lung cancer. 3-Deazaneplanocin-A (DZNep, an inhibitor of EZH2, up-regulates the expression of genes silenced by H3K27me3. In this study we compared the in vitro antineoplastic activity of different inhibitors of EZH2; DZNep, U-4149 and Gsk-126, alone and in combination with 5-Aza-CdR, on the human A549 lung adenocarcinoma cells. U-4149, an analogue of DZNep, was more potent than either DZNep or Gsk-126. The reduction in colony formation was dose- and time-dependent for each EZH2 inhibitors. Combination treatment of 5-Aza-CdR with the EZH2 inhibitors showed a synergistic antineoplastic activity. 5-Aza-CdR and U-4149 was the most potent combination. The in vitro antineoplastic activity of these agents was evaluated by inhibition of growth, colony formation, induction of senescence and apoptosis. All the drug combinations induced signs of senescence and apoptosis. Analysis by gene expression by qRT-PCR showed that the combinations increased the expression of several TSGs to a greater extent that either agent alone. In conclusion, epigenetic therapy that specifically targets DNA and histone methylation has interesting potential for the treatment of lung cancer and merits further investigation.

  5. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases

    DEFF Research Database (Denmark)

    Johansson, K.; Ramaswamy, S.; Ljungcrantz, C.

    2001-01-01

    Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides and activate a number of medically important nucleoside analogs. Here we report the structure of the Drosophila deoxyribonucleoside kinase with deoxycytidine bound at the nucleoside binding site and that of the human deoxyguanosine ki......; this is apparently due to the presence of Arg 118, which provides favorable hydrogen bonding interactions with the substrate. The two new structures provide an explanation for the substrate specificity of cellular deoxyribonucleoside kinases....

  6. Deoxycytidine transport and metabolism in choroid plexus

    Energy Technology Data Exchange (ETDEWEB)

    Spector, R.; Huntoon, S.

    1983-05-01

    In vitro, the transport into and release of (/sup 3/H)deoxycytidine from the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, were studied separately. By use of the ability of nitrobenzylthioinosine (NBTI) to inhibit deoxycytidine efflux from choroid plexus, the transport of 1 microM (/sup 3/H)deoxycytidine into choroid plexus at 37 degrees C was measured. Deoxycytidine was transported into choroid plexus against a concentration gradient by a saturable process that depended on intracellular energy production, but not intracellular binding or metabolism. The Michaelis-Menten constant (KT) for the active transport of deoxycytidine into choroid plexus was 15 microM. The active transport system for deoxycytidine was inhibited by naturally occurring nucleosides and deoxynucleosides, but not by 1 mM probenecid and 2-deoxyribose or 100 microM cytosine and cytosine arabinoside. With less than 1 microM (/sup 3/H)deoxycytidine in the medium, the choroid plexus accumulated (/sup 3/H)deoxycytidine against a concentration gradient. However, approximately 50% of the (/sup 3/H)deoxycytidine was phosphorylated to (/sup 3/H)deoxycytidine nucleotides at a low extracellular (/sup 3/H)deoxycytidine concentration (6 nM) in 15-min incubations. This accumulation process depended, in part, on saturable intracellular phosphorylation. These studies provide further evidence that the choroid plexus contains an active nucleoside transport system of low specificity for deoxynucleosides and ribonucleosides, and a separate, saturable efflux system for deoxynucleosides which is very sensitive to inhibition by NBTI.

  7. Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)-like group vary significantly in substrate specificity, kinetics and feed-back regulation.

    Science.gov (United States)

    Knecht, Wolfgang; Petersen, Gitte Ebert; Munch-Petersen, Birgitte; Piskur, Jure

    2002-01-25

    In eukaryotic cells deoxyribonucleoside kinases belonging to three phylogenetic sub-families have been found: (i) thymidine kinase 1 (TK1)-like enzymes, which are strictly pyrimidine deoxyribonucleoside-specific kinases; (ii) TK2-like enzymes, which include pyrimidine deoxyribonucleoside kinases and a single multisubstrate kinase from Drosophila melanogaster (Dm-dNK); and (iii) deoxycytidine/deoxyguanosine kinase (dCK/dGK)-like enzymes, which are deoxycytidine and/or purine deoxyribonucleoside-specific kinases. We cloned and characterized two new deoxyribonucleoside kinases belonging to the TK2-like group from the insect Bombyx mori and the amphibian Xenopus laevis. The deoxyribonucleoside kinase from B. mori (Bm-dNK) turned out to be a multisubstrate kinase like Dm-dNK. But uniquely for a deoxyribonucleoside kinase, Bm-dNK displayed positive cooperativity with all four natural deoxyribonucleoside substrates. The deoxyribonucleoside kinase from X. laevis (Xen-PyK) resembled closely the human and mouse TK2 enzymes displaying their characteristic Michaelis-Menten kinetic with deoxycytidine and negative cooperativity with its second natural substrate thymidine. Bm-dNK, Dm-dNK and Xen-PyK were shown to be homodimers. Significant differences in the feedback inhibition by deoxyribonucleoside triphosphates between these three enzymes were found. The insect multisubstrate deoxyribonucleoside kinases Bm-dNK and Dm-dNK were only inhibited by thymidine triphosphate, while Xen-PyK was inhibited by thymidine and deoxycytidine triphosphate in a complex pattern depending on the deoxyribonucleoside substrate. The broad substrate specificity and different feedback regulation of the multisubstrate insect deoxyribonucleoside kinases may indicate that these enzymes have a different functional role than the other members of the TK2-like group. Copyright 2002 Academic Press.

  8. Crystal Structure of Human Nicotinamide Riboside Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Khan,J.; Xiang, S.; Tong, L.

    2007-01-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD{sup +} as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 {angstrom} resolution and in a ternary complex with ADP and tiazofurin at 2.7 {angstrom} resolution. The active site is located in a groove between the central parallel {beta} sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  9. Crystal structure of human nicotinamide riboside kinase.

    Science.gov (United States)

    Khan, Javed A; Xiang, Song; Tong, Liang

    2007-08-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  10. Deoxyribonucleoside kinases: two enzyme families catalyze the same reaction.

    Science.gov (United States)

    Sandrini, Michael P B; Piskur, Jure

    2005-05-01

    Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kinases can phosphorylate and thereby activate a variety of anti-cancer and antiviral prodrugs. Recently, the crystal structure of human TK1 has been solved and has revealed that enzymes with fundamentally different origins and folds catalyze similar, crucial cellular reactions.

  11. Sensitization of cells to x-ray by 5-chloro-2'-deoxycytidine: studies on the metabolism and incorporation in DNA of a novel radiosensitizer

    Energy Technology Data Exchange (ETDEWEB)

    Perez, L.M.

    1985-01-01

    Rapid catabolism and generalized toxicity have limited the use of 5-halogenated deoxyuridine analogs as tumor radiosensitizers. This problem was approached by utilizing a deoxycytidine analog, 5-chloro-2'-deoxycytidine (CldC) coadministered with tetrahydrouridine (H/sub 4/U), an inhibitor of its deamination. Enzyme kinetic data and studies in various cell lines indicate that CldC + H/sub 4/U may be metabolized as follows: CldC ..-->.. CldCMP ..-->.. CldUMP ..-->.. ..-->.. CldUTP ..-->.. DNA ((1) deoxycytidine kinase; (2) deoxycytidylate deaminase dCMPD; (3) thymidylate kinase; and (4) DNA polymerase). All of these enzymes are elevated in human and mouse malignant tumors above that of normal tissues; especially dCMPD, which is elevated 20- to 80-fold. In tumors high in cytidine deaminase (CD), the following pathway is also likely involved: CldC ..-->.. CldU ..-->.. CldUMP ..-->.. ..-->.. CldUTP ..-->.. DNA ((1) Cd and (2) thymidine kinase). It is not known whether one of these two pathways (Cd ..-->.. TK or dCK ..-->.. dCMPD) is preferred in tumors. CldC + H/sub 4/U sensitized cells (HEp-2, S-180 and RIF-1) to X-ray and was incorporated in DNA as CldU at a substitution level of 20 to 30%. CHO cells, which intrinsically lack both CD and dCMPD were sensitized by CldU but displayed only marginal X-ray sensitization with CldC + H/sub 4/U. Thus, both pathways of conversion (dCK ..-->.. dCMPD; CD ..-->.. TK) of CldC to CldUMP cannot be blocked.

  12. Signaling of mechanical stretch in human keratinocytes via MAP kinases.

    Science.gov (United States)

    Kippenberger, S; Bernd, A; Loitsch, S; Guschel, M; Müller, J; Bereiter-Hahn, J; Kaufmann, R

    2000-03-01

    Cells within human skin are permanently exposed to mechanical stretching. Here we present evidence that alterations in cell shape trigger biochemical signaling via MAP kinases in human keratinocytes. In an in vitro attempt we demonstrate a fast but transient activation of extracellular signal-regulated kinases 1/2 in response to cell stretch. This activation is reversed by preincubation with functional blocking antibodies directed towards beta1-integrins. As a second member of MAP kinases, stress-activated protein kinase/c-JUN NH2-terminal kinase was activated in a slower fashion, peaking at 1 h after the initial stimulus. The delay in signal transmission suggests that extracellular signal-regulated kinases 1/2 and stress-activated protein kinase/c-JUN NH2-terminal kinase do not share the same signaling pathway. p38 was not activated by cell stretching. The contribution of cytoskeletal elements in signal perception and transduction was evaluated by selective disruption of either actin filaments, microtubules, or keratin filaments but showed no clear effect on stretch-induced activation of extracellular signal-regulated kinases 1/2 and stress-activated protein kinase/c-JUN NH2-terminal kinase. In conclusion we found evidence of a cell-shape-dependent activation of MAP kinases in human keratinocytes disclosing beta1-integrins as putative mechano-transducers. It is likely that alterations of skin mechanics in vivo underlying pathogenic processes like wound formation and healing trigger physiologic responses via the MAP kinase pathway.

  13. Global demethylation of rat chondrosarcoma cells after treatment with 5-aza-2'-deoxycytidine results in increased tumorigenicity.

    Directory of Open Access Journals (Sweden)

    Christopher A Hamm

    Full Text Available Abnormal patterns of DNA methylation are observed in several types of human cancer. While localized DNA methylation of CpG islands has been associated with gene silencing, the effect that genome-wide loss of methylation has on tumorigenesis is not completely known. To examine its effect on tumorigenesis, we induced DNA demethylation in a rat model of human chondrosarcoma using 5-aza-2-deoxycytidine. Rat specific pyrosequencing assays were utilized to assess the methylation levels in both LINEs and satellite DNA sequences following 5-aza-2-deoxycytidine treatment. Loss of DNA methylation was accompanied by an increase in invasiveness of the rat chondrosarcoma cells, in vitro, as well as by an increase in tumor growth in vivo. Subsequent microarray analysis provided insight into the gene expression changes that result from 5-aza-2-deoxycytidine induced DNA demethylation. In particular, two genes that may function in tumorigenesis, sox-2 and midkine, were expressed at low levels in control cells but upon 5-aza-2-deoxycytidine treatment these genes became overexpressed. Promoter region DNA analysis revealed that these genes were methylated in control cells but became demethylated following 5-aza-2-deoxycytidine treatment. Following withdrawal of 5-aza-2-deoxycytidine, the rat chondrosarcoma cells reestablished global DNA methylation levels that were comparable to that of control cells. Concurrently, invasiveness of the rat chondrosarcoma cells, in vitro, decreased to a level indistinguishable to that of control cells. Taken together these experiments demonstrate that global DNA hypomethylation induced by 5-aza-2-deoxycytidine may promote specific aspects of tumorigenesis in rat chondrosarcoma cells.

  14. Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

    Directory of Open Access Journals (Sweden)

    Anamika Krishanpal

    2009-12-01

    Full Text Available Abstract Background Protein kinases are involved in diverse spectrum of cellular processes. Availability of draft version of the human genomic data in the year 2001 enabled recognition of repertoire of protein kinases. However, over the years the human genomic data is being refined and the current release of human genomic data has helped us to recognize a larger repertoire of over 900 human protein kinases represented mainly by splice variants. Results Many of these identified protein kinases are alternatively spliced products. Interestingly, some of the human kinase splice variants appear to be significantly diverged in terms of their functional properties as represented by incorporation or absence of one or more domains. Many sets of protein kinase splice variants have substantially different domain organization and in a few sets of splice variants kinase domains belong to different subfamilies of kinases suggesting potential participation in different signal transduction pathways. Conclusions Addition or deletion of a domain between splice variants of multi-domain kinases appears to be a means of generating differences in the functional features of otherwise similar kinases. It is intriguing that marked sequence diversity within the catalytic regions of some of the splice variant kinases result in kinases belonging to different subfamilies. These human kinase splice variants with different functions might contribute to diversity of eukaryotic cellular signaling.

  15. Structures of thymidine kinase 1 of human and mycoplasma origin

    DEFF Research Database (Denmark)

    Welin, Martin; Kosinska, Urszula; Mikkelsen, Nils-Egil

    2004-01-01

    Cytosolic thymidine kinase, TK1, is a well-known cell cycle regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs as AZT. We have now determined the first structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in com...... differs fundamentally from the structures of the other deoxyribonucleoside kinases indicating a different evolutionary origin....

  16. Acquired pyruvate kinase deficiency. The effect of maleic acid upon human erythrocyte pyruvate kinase

    NARCIS (Netherlands)

    Sprengers, E.D.; Staal, Gerard E.J.

    1979-01-01

    1. 1. Maleic acid is shown to be able to bind the thiol compound 2-mercaptoethanol. This is fully consistent with the data of Morgan and Friedman (1938). 2. 2. Human erythrocyte pyruvate kinase dissolved and quantitated in Trismaleate shows a loss of positive homotropic interactions, as compared

  17. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...

  18. Human UMP-CMP kinase 2, a novel nucleoside monophosphate kinase localized in mitochondria.

    Science.gov (United States)

    Xu, Yunjian; Johansson, Magnus; Karlsson, Anna

    2008-01-18

    Enzyme deficiency in the salvage pathway of deoxyribonucleotide synthesis in mitochondria can cause mtDNA depletion syndromes. We have identified a human mitochondrial UMP-CMP kinase (UMP-CMPK, cytidylate kinase; EC 2.7.4.14), designated as UMP-CMP kinase 2 (UMP-CMPK2). The C-terminal domain of this 449-amino acid protein contains all consensus motifs of a nucleoside monophosphate kinase. Phylogenetic analysis showed that UMP-CMPK2 belonged to a novel nucleoside monophosphate kinase family, which was closer to thymidylate kinase than to cytosolic UMP-CMP kinase. Subcellular localization with green fluorescent protein fusion proteins illustrated that UMP-CMPK2 was localized in the mitochondria of HeLa cells and that the mitochondrial targeting signal was included in the N-terminal 22 amino acids. The enzyme was able to phosphorylate dUMP, dCMP, CMP, and UMP with ATP as phosphate donor, but the kinetic properties were different compared with the cytosolic UMP-CMPK. Its efficacy to convert dUMP was highest, followed by dCMP, whereas CMP and UMP were the poorest substrates. It also phosphorylated the monophosphate forms of the nucleoside analogs ddC, dFdC, araC, BVDU, and FdUrd, which suggests that UMP-CMPK2 may be involved in mtDNA depletion caused by long term treatment with ddC or other pyrimidine analogs. UMP-CMPK2 mRNA expression was exclusively detected in chronic myelogenous leukemia K-562 and lymphoblastic leukemia MOLT-4 among eight studied cancer cell lines. Particular high expression in leukemia cells, dominant expression in bone marrow, and tight correlation with macrophage activation and inflammatory response suggest that UMP-CMPK2 may have other functions in addition to the supply of substrates for mtDNA synthesis.

  19. Mutation Study of Two Thymidine Kinases 

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Munch-Petersen, Birgitte; Eklund, Hans

    The deoxyribonucleoside kinase, thymidine kinase 1 (TK1), is a key enzyme in the salvage pathway, where it catalyzes the first of three phosphate transfers from ATP to thymidine. Besides from their importance in DNA metabolism deoxyribonucleoside kinases are also important for activation of antic...... not phosphorylate the anticancer analog 1-β-D-arabinofuranosylcytosine (AraC), however. The HuTK1 mutant has been crystallized, and azidothymidine monophosphate has been modelled into the active site.......The deoxyribonucleoside kinase, thymidine kinase 1 (TK1), is a key enzyme in the salvage pathway, where it catalyzes the first of three phosphate transfers from ATP to thymidine. Besides from their importance in DNA metabolism deoxyribonucleoside kinases are also important for activation...... of anticancer and antiviral nucleoside pro-drugs. Humans have four different deoxyribonucleoside kinases, the two cytosolic: TK1 and deoxycytidine kinase (dCK) and the two mitochondrial: thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK). In insects, a single gene encodes a multi substrate kinase...

  20. Structural comparison of human mammalian ste20-like kinases.

    Directory of Open Access Journals (Sweden)

    Christopher J Record

    2010-08-01

    Full Text Available The serine/threonine mammalian Ste-20 like kinases (MSTs are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains.We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family.The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  1. Mitogen-activated protein kinase kinase 4 (MAP2K4 promotes human prostate cancer metastasis.

    Directory of Open Access Journals (Sweden)

    Janet M Pavese

    Full Text Available Prostate cancer (PCa is the second leading cause of cancer death in the US. Death from PCa primarily results from metastasis. Mitogen-activated protein kinase kinase 4 (MAP2K4 is overexpressed in invasive PCa lesions in humans, and can be inhibited by small molecule therapeutics that demonstrate favorable activity in phase II studies. However, MAP2K4's role in regulating metastatic behavior is controversial and unknown. To investigate, we engineered human PCa cell lines which overexpress either wild type or constitutive active MAP2K4. Orthotopic implantation into mice demonstrated MAP2K4 increases formation of distant metastasis. Constitutive active MAP2K4, though not wild type, increases tumor size and circulating tumor cells in the blood and bone marrow. Complementary in vitro studies establish stable MAP2K4 overexpression promotes cell invasion, but does not affect cell growth or migration. MAP2K4 overexpression increases the expression of heat shock protein 27 (HSP27 protein and protease production, with the largest effect upon matrix metalloproteinase 2 (MMP-2, both in vitro and in mouse tumor samples. Further, MAP2K4-mediated increases in cell invasion are dependent upon heat shock protein 27 (HSP27 and MMP-2, but not upon MAP2K4's immediate downstream targets, p38 MAPK or JNK. We demonstrate that MAP2K4 increases human PCa metastasis, and prolonged over expression induces long term changes in cell signaling pathways leading to independence from p38 MAPK and JNK. These findings provide a mechanistic explanation for human studies linking increases in HSP27 and MMP-2 to progression to metastatic disease. MAP2K4 is validated as an important therapeutic target for inhibiting human PCa metastasis.

  2. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    Energy Technology Data Exchange (ETDEWEB)

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen (Sanofi); (Michigan); (Texas)

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  3. A multisubstrate deoxyribonucleoside kinase from plants.

    Science.gov (United States)

    Clausen, Anders Ranegaard; Girandon, Lenart; Knecht, Wolfgang; Survery, Sabeen; Andreasson, Erik; Munch-Petersen, Birgitte; Piskur, Jure

    2008-01-01

    Deoxyribonucleoside kinases catalyze the rate limiting step during the salvage of deoxyribonucleosides and convert them into the corresponding monophosphate compounds. We have identified and characterized a unique multisubstrate deoxyribonucleoside kinase from plants. The phylogenetic relationship and biochemical properties suggest that this deoxyribonucleoside kinase represents a living fossil resembling the progenitor of the modern animal deoxycytidine, deoxyguanosine and thymidine 2 kinases. The broad substrate specificity makes this enzyme an interesting candidate to be evaluated as a suicide gene in anti-cancer therapy.

  4. Deoxyribonucleoside kinases: two enzyme families catalyze the same reaction

    DEFF Research Database (Denmark)

    Sandrini, Michael; Piskur, Jure

    2005-01-01

    Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kin...

  5. Crystal structures of human pyridoxal kinase in complex with the neurotoxins, ginkgotoxin and theophylline: insights into pyridoxal kinase inhibition.

    Science.gov (United States)

    Gandhi, Amit K; Desai, Jigar V; Ghatge, Mohini S; di Salvo, Martino L; Di Biase, Stefano; Danso-Danquah, Richmond; Musayev, Faik N; Contestabile, Roberto; Schirch, Verne; Safo, Martin K

    2012-01-01

    Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B₆, pyridoxal 5'-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B₆ is converted to PLP by PL kinase. PLP is the B₆ vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B₆, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects.

  6. Crystal structures of human pyridoxal kinase in complex with the neurotoxins, ginkgotoxin and theophylline: insights into pyridoxal kinase inhibition.

    Directory of Open Access Journals (Sweden)

    Amit K Gandhi

    Full Text Available Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase, resulting in deficiency of the active cofactor form of vitamin B₆, pyridoxal 5'-phosphate (PLP. Pyridoxal (PL, an inactive form of vitamin B₆ is converted to PLP by PL kinase. PLP is the B₆ vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B₆, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects.

  7. Crystal Structures of Human Pyridoxal Kinase in Complex with the Neurotoxins, Ginkgotoxin and Theophylline: Insights into Pyridoxal Kinase Inhibition

    Science.gov (United States)

    Ghatge, Mohini S.; di Salvo, Martino L.; Di Biase, Stefano; Danso-Danquah, Richmond; Musayev, Faik N.; Contestabile, Roberto; Schirch, Verne; Safo, Martin K.

    2012-01-01

    Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B6, pyridoxal 5′-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B6 is converted to PLP by PL kinase. PLP is the B6 vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B6, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects. PMID:22879864

  8. Enantioselectivity of human AMP, dTMP and UMP-CMP kinases.

    Science.gov (United States)

    Alexandre, Julie A C; Roy, Béatrice; Topalis, Dimitri; Pochet, Sylvie; Périgaud, Christian; Deville-Bonne, Dominique

    2007-01-01

    L-nucleoside analogues such as lamivudine are active for treating viral infections. Like D-nucleosides, the biological activity of the L-enantiomers requires their stepwise phosphorylation by cellular or viral kinases to give the triphosphate. The enantioselectivity of NMP kinases has not been thoroughly studied, unlike that of deoxyribonucleoside kinases. We have therefore investigated the capacity of L-enantiomers of some natural (d)NMP to act as substrates for the recombinant forms of human uridylate-cytidylate kinase, thymidylate kinase and adenylate kinases 1 and 2. Both cytosolic and mitochondrial adenylate kinases were strictly enantioselective, as they phosphorylated only D-(d)AMP. L-dTMP was a substrate for thymidylate kinase, but with an efficiency 150-fold less than D-dTMP. Both L-dUMP and L-(d)CMP were phosphorylated by UMP-CMP kinase although much less efficiently than their natural counterparts. The stereopreference was conserved with the 2'-azido derivatives of dUMP and dUMP while, unexpectedly, the 2'-azido-D-dCMP was a 4-fold better substrate for UMP-CMP kinase than was CMP. Docking simulations showed that the small differences in the binding of D-(d)NMP to their respective kinases could account for the differences in interactions of the L-isomers with the enzymes. This in vitro information was then used to develop the in vivo activation pathway for L-dT.

  9. Inhibition of thymidylate synthase by 2',2'-difluoro-2'-deoxycytidine (Gemcitabine) and its metabolite 2',2'-difluoro-2'-deoxyuridine.

    Science.gov (United States)

    Honeywell, Richard J; Ruiz van Haperen, Veronique W T; Veerman, Gijsbert; Smid, Kees; Peters, Godefridus J

    2015-03-01

    2',2'-Difluoro-2'-deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2',2'-difluoro-2'-deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of [5-(3)H]-2'-deoxyuridine or [5-(3)H]-2'-deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4h exposure to 1 μM dFdC tritium release was inhibited by 50% but did not increase after 24h, Inhibition was also observed following dFdU at 100 μM. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase. Inhibition of the enzyme resulted in a relative increase of mis-incorporation of [5-(3)H]-2'-deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However

  10. Identification and characterization of Nek6 protein kinase, a potential human homolog of NIMA histone H3 kinase.

    Science.gov (United States)

    Hashimoto, Yoshihiro; Akita, Hidetoshi; Hibino, Mitsunobu; Kohri, Kenjiro; Nakanishi, Makoto

    2002-05-03

    In Aspergillus nidulans, the kinase activity of NIMA (never in mitosis, gene A) is critical for the initiation of mitosis. NIMA regulates mitotic chromatin condensation through phosphorylation of histone H3 at serine 10. In the present study, we identified human Nek6 (hNek6), a member of the mammalian NIMA-related kinases. The predicted hNek6 protein is comprised of 338 amino acids. Northern blot analysis revealed that hNek6 transcripts are ubiquitously expressed with the highest expression found in the heart and skeletal muscle. Lower cell cycle-dependent expression of hNek6 transcripts was observed in the early G1 phase. GFP-fused hNek6 protein showed both nuclear and cytoplasmic localizations in HeLa cells. Fluorescence in situ hybridization using full-length hNek6 cDNA as a probe showed that the hNek6 gene is localized to human chromosome 9q33-34, a region at which the loss of heterozygosity is associated with transitional cell carcinomas. Importantly, recombinant hNek6 protein produced in insect cells effectively phosphorylated histones H1 and H3, but not casein. Thus, these results suggest that, unlike other mammalian NIMA-related kinases, Nek6 is a mitotic histone kinase which regulates chromatin condensation in mammalian cells. Copyright 2002 Elsevier Science (USA).

  11. Casein kinase II is elevated in solid human tumours and rapidly proliferating non-neoplastic tissue

    DEFF Research Database (Denmark)

    Münstermann, U; Fritz, G; Seitz, G

    1990-01-01

    Protein kinase CKII (i.e. casein kinase II, CKII, NII) is expressed at a higher level in rapidly proliferating tissues and in solid human tumours (e.g. colorectal carcinomas) when compared to the corresponding non-neoplastic colorectal mucosa. This could be shown by (a) Western blotting of cellular...

  12. [Reversion transcriptional expression of DAPK in bladder cancer T24 cells 5-aza-2'-deoxycytidine].

    Science.gov (United States)

    Xu, Ning-ru; Liu, Chun-xiao; Zheng, Shao-bo; Li, Hu-lin; Xu, Ya-wen; Xu, Kai

    2009-09-01

    To investigate the methylation status of the promoter of resion death associated protein kinase (DAPK) gene in bladder cancer cell (T24), and study the effect of 5-aza-2'-deoxycytidine (5-aza-dc) on DAPK gene reactive expression in T24 and its inhibitory effect on T24. The bladder cancer cell T24 was treated with different doses of 5-aza-dc. The inhibitory effect and apoptosis rate were detected by MTT and flow cytometry, and the changes of DAPK mRNA and protein expression and the methylation status of DAPK promoter were assessed by RT-PCR, Western blotting, and methylation specific PCR, respectively. The growth of bladder cancer cell was inhibited significantly and the maximal apoptosis rate detected by flow cytometry was (24.12-/+1.4)%. DAPK mRNA was not expressed in bladder cancer cell T24 in normal conditions. DAPK mRNA and protein re-expressed after 5-aza-dc (12.5 micromol/L) treatment in cell line T24 for 24 h, and DAPK promoter became unmethylated. The promoter methylation can be an important factor for silencing the expression of DAPK in bladder cancer cell. 5-aza-dc can inhibit the growth and induce apoptosis of bladder cancer cells through reversing unmethylation status of DAPK promoter.

  13. wKinMut-2: Identification and Interpretation of Pathogenic Variants in Human Protein Kinases

    DEFF Research Database (Denmark)

    Vazquez, Miguel; Pons, Tirso; Brunak, Søren

    2016-01-01

    forest approach. To understand the biological mechanisms causative of human diseases and cancer, information from pertinent reference knowledgebases and the literature is automatically mined, digested and homogenized. Variants are visualized in their structural contexts and residues affecting catalytic...... is often scattered across different sources, which makes the integrative analysis complex and laborious. wKinMut-2 constitutes a solution to facilitate the interpretation of the consequences of human protein kinase variation. Nine methods predict their pathogenicity, including a kinase-specific random...

  14. Src-family tyrosine kinase activities are essential for differentiation of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Xiong Zhang

    2014-11-01

    Full Text Available Embryonic stem (ES cells are characterized by pluripotency, defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade, great progress has been made on the cell culture conditions, transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions, responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein–tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells, and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal, while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation, the role of this kinase family in human ES cells is largely unknown. Here, we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome, Fyn, c-Yes, c-Src, Lyn, Lck and Hck were expressed in H1, H7 and H9 hES cells, while Fgr, Blk, Srm, Brk, and Frk transcripts were not detected. Of these, c-Yes, Lyn, and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs, while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast, Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation, cultures were treated with inhibitors specific for the Src kinase family. Remarkably, human ES cells maintained in the presence of the potent

  15. Origin of increased deoxycytidine excretion into urine of rats bearing Yoshida ascites sarcoma

    Energy Technology Data Exchange (ETDEWEB)

    Shimizu, M.; Fujimura, S.

    1984-06-01

    The metabolism of deoxycytidine (dCyd) and dCyd nucleotides in Yoshida ascites sarcoma (YS) cells and the host rat liver was investigated with reference to the increased excretion of urinary dCyd. Incorporation of (/sup 14/C)orotic acid into the livers of rats at the fifth day after the transplantation of YS cells, was 2 times higher than that into the normal rat livers. After the injection of (/sup 14/C)orotic acid, the ratio of the specific radioactivity of cytidylate to uridylate moieties of the host liver RNA was measured and found to be higher than that of normal rat liver RNA and to be similar to that of YS cell RNA. When (/sup 14/C)orotic acid was injected into rats followed by the transplantation of YS cells, the radioactivities present in the livers disappeared more rapidly than those in the control rat livers. The activities of pyrimidine de novo synthesis enzymes, such as cytidine triphosphate synthetase and cytidine diphosphate reductase, in YS were higher than those in both rat ascites hepatoma AH 7974 and Walker 256 carcinosarcoma, the transplantations of which did not induce increased excretion of dCyd into urine of the hosts. The activities of dCyd kinase and dCyd deaminase in YS cells were lower than those in the other two tumors investigated. The activities of cytidine triphosphate synthetase and cytidine diphosphate reductase in the livers of YS-bearing rats were elevated compared with those in the livers of rat ascites hepatoma AH 7974- or Walker 256 carcinosarcoma-bearing rats and normal rats, while the activities of dCyd kinase, 5'-nucleotidase, and dCyd deaminase were similar between normal rat livers and tumor-bearing rat livers.

  16. Nek8, a NIMA family kinase member, is overexpressed in primary human breast tumors.

    Science.gov (United States)

    Bowers, Alex J; Boylan, John F

    2004-03-17

    The family of human Nek (NIMA Related Kinase) kinases currently contains 11 members. We have identified Nek8 as a new member of the Nek kinase family. For many of the Nek family members, primary tumor expression data and function have been limited. However, all of the Nek family proteins share considerable homology with the Never In Mitosis, gene A (NIMA) kinase from the filamentous fungus Aspergillus nidulans. NIMA, as well as its most closely related human ortholog, Nek2, are required for G(2)/M progression and promote centrosome maturation during mitosis. We isolated Nek8 from a primary human colon cDNA library, and found it to be highly homologous to murine Nek8. Recently, a previously named Nek8 sequence was renamed Nek9/Nercc1 in Genbank due to its lack of homology to murine Nek8 and its high homology to murine Nek9. Interestingly, in our study, phylogenetic analysis suggests that human Nek8 and Nek9 form a subfamily within the Nek family. Nek8 has high homology to the Nek family kinase domain as well as to a regulator of chromosome condensation domain (RCC1), which is also present in Nek9. The open reading frame of human Nek8 encodes a 692 amino-acid protein with a calculated molecular weight of 75 kDa. Nek8 is differently expressed between normal human breast tissue and breast tumors. Overexpression of a mutated kinase domain Nek8 in U2-0S cells led to a decrease in actin protein, and a small increase in the level of cdk1/cyclinB1. Our data demonstrate for the first time that Nek8 is a novel tumor associated gene, and shares considerable sequence homology with the Nek family of protein kinases and may be involved in G(2)/M progression.

  17. Assignment of casein kinase 2 alpha sequences to two different human chromosomes

    DEFF Research Database (Denmark)

    Boldyreff, B; Klett, C; Göttert, E

    1992-01-01

    Human casein kinase 2 alpha gene (CK-2-alpha) sequences have been localized within the human genome by in situ hybridization and somatic cell hybrid analysis using a CK-2 alpha cDNA as a probe. By in situ hybridization, the CK-2 alpha cDNA could be assigned to two different loci, one on 11p15.1-ter...

  18. Differential inhibitor sensitivity between human kinases VRK1 and VRK2.

    Directory of Open Access Journals (Sweden)

    Marta Vázquez-Cedeira

    Full Text Available Human vaccinia-related kinases (VRK1 and VRK2 are atypical active Ser-Thr kinases implicated in control of cell cycle entry, apoptosis and autophagy, and affect signalling by mitogen activated protein kinases (MAPK. The specific structural differences in VRK catalytic sites make them suitable candidates for development of specific inhibitors. In this work we have determined the sensitivity of VRK1 and VRK2 to kinase inhibitors, currently used in biological assays or in preclinical studies, in order to discriminate between the two proteins as well as with respect to the vaccinia virus B1R kinase. Both VRK proteins and vaccinia B1R are poorly inhibited by inhibitors of different types targeting Src, MEK1, B-Raf, JNK, p38, CK1, ATM, CHK1/2 and DNA-PK, and most of them have no effect even at 100 µM. Despite their low sensitivity, some of these inhibitors in the low micromolar range are able to discriminate between VRK1, VRK2 and B1R. VRK1 is more sensitive to staurosporine, RO-31-8220 and TDZD8. VRK2 is more sensitive to roscovitine, RO 31-8220, Cdk1 inhibitor, AZD7762, and IC261. Vaccinia virus B1R is more sensitive to staurosporine, KU55933, and RO 31-8220, but not to IC261. Thus, the three kinases present a different pattern of sensitivity to kinase inhibitors. This differential response to known inhibitors can provide a structural framework for VRK1 or VRK2 specific inhibitors with low or no cross-inhibition. The development of highly specific VRK1 inhibitors might be of potential clinical use in those cancers where these kinases identify a clinical subtype with a poorer prognosis, as is the case of VRK1 in breast cancer.

  19. Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

    Science.gov (United States)

    Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

    2017-10-01

    Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

  20. Molecular cloning and characterization of a novel human kinase ...

    Indian Academy of Sciences (India)

    Ensembl Genome Browser located PDIK1L to human chromosome 1p35.3. It spans about 13.7 kb and consists of four exons and three introns. Multiple-tissue cDNA panel PCR revealed that the gene is expressed widely in human tissues: liver, kidney, pancreas, spleen, thymus and prostate. The protein appears to be ...

  1. Low-density crystal packing of human protein kinase CK2 catalytic subunit in complex with resorufin or other ligands

    DEFF Research Database (Denmark)

    Klopffleisch, Karsten; Niefind, Karsten; Issinger, Olaf-Georg

    2012-01-01

    A low-resolution structure of the catalytic subunit CK2α of human protein kinase CK2 (formerly known as casein kinase 2) in complex with the ATP-competitive inhibitor resorufin is presented. The structure supplements previous human CK2α structures in which the interdomain hinge/helix αD region...

  2. Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

    DEFF Research Database (Denmark)

    Grankowski, N; Boldyreff, B; Issinger, O G

    1991-01-01

    cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...

  3. The Effect of Creatine Kinase Inhibition on Contractile Properties of Human Resistance Arteries

    NARCIS (Netherlands)

    Taherzadeh, Zhila; Karamat, Fares A.; Ankum, Willem M.; Clark, Joseph F.; van Montfrans, Gert A.; van Bavel, Ed; Brewster, Lizzy M.

    2016-01-01

    Creatine kinase (CK) is a main predictor of blood pressure, and this is thought to largely depend on high resistance artery contractility. We previously reported an association between vascular contractility and CK in normotensive pregnancy, but pregnancy is a strong CK inducer, and data on human

  4. Salinomycin causes migration and invasion of human fibrosarcoma cells by inducing MMP-2 expression via PI3-kinase, ERK-1/2 and p38 kinase pathways.

    Science.gov (United States)

    Yu, Seon-Mi; Kim, Song Ja

    2016-06-01

    Salinomycin (SAL) is a polyether ionophore antibiotic that has recently been shown to regulate a variety of cellular responses in various human cancer cells. However, the effects of SAL on metastatic capacity of HT1080 human fibrosarcoma cells have not been elucidated. We investigated the effect of SAL on migration and invasion, with emphasis on the expression and activation of matrix metalloproteinase (MMP)-2 in HT1080 human fibrosarcoma cells. Treatment of SAL promoted the expression and activation of MMP-2 in a dose- and time-dependent manner, as detected by western blot analysis, gelatin zymography, and real-time polymerase chain reaction. SAL also increased metastatic capacities, as determined by an increase in the migration and invasion of cells using the wound healing assay and the invasion assay, respectively. To confirm the detailed molecular mechanisms of these effects, we measured the activation of phosphoinositide 3 kinase (PI3-kinase) and mitogen-activated protein kinase (MAPK)s (ERK-1/2 and p38 kinase), as detected by the phosphorylated proteins through western blot analysis. SAL treatment increased the phosphorylation of Akt and MAPKs. Inhibition of PI3-kinase, ERK-1/2, and p38 kinase with LY294002, PD98059, and SB203580, respectively, in the presence of SAL suppressed the metastatic capacity by reducing MMP-2 expression, as determined by gelatin zymography. Our results indicate that the PI3-kinase and MAPK signaling pathways are involved in migration and invasion of HT1080 through induction of MMP-2 expression and activation. In conclusion, SAL significantly increases the metastatic capacity of HT1080 cells by inducing MMP-2 expression via PI3-kinase and MAPK pathways. Our results suggest that SAL may be a potential agent for the study of cancer metastatic capacities.

  5. Ser2 is the autophosphorylation site in the beta subunit from bicistronically expressed human casein kinase-2 and from native rat liver casein kinase-2 beta

    DEFF Research Database (Denmark)

    Boldyreff, B; James, P; Staudenmann, W

    1993-01-01

    Human casein kinase-2 (CK-2) subunits alpha and beta were bicistronically expressed in bacteria. The recombinant holoenzyme shared all investigated properties with the native CK-2 from mammalian sources (rat liver, Krebs II mouse ascites tumour cells). Contrary to recombinant human CK-2 produced...

  6. Isolation of a human lymphoblastoid line heterozygous at the thymidine kinase locus: possibility for a rapid human cell mutation assay

    Energy Technology Data Exchange (ETDEWEB)

    Skopek, T.R.; Liber, H.L.; Penman, B.W.; Thilly, W.G.

    1978-09-29

    A thymidine kinase heterozygote designated H2BT has been isolated from the human lymphoblast line HH4. Significant increase in the trifluorothymidine-resistant fraction was observed in the new cell line following treatment with the mutagens ICR-191 and butylmethansulfonate. Phenotypic expression was complete forty-eight hours after treatment.

  7. Structure and Substrate Recruitment of the Human Spindle Checkpoint Kinase Bub1

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Jungseog; Yang, Maojun; Li, Bing; Qi, Wei; Zhang, Chao; Shokat, Kevan M.; Tomchick, Diana R.; Machius, Mischa; Yu, Hongtao (UCSF); (UTSMC)

    2009-11-10

    In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.

  8. Phosphatidylcholine metabolism and choline kinase in human osteoblasts.

    Science.gov (United States)

    Li, Zhuo; Wu, Gengshu; van der Veen, Jelske N; Hermansson, Martin; Vance, Dennis E

    2014-06-01

    There is a paucity of information about phosphatidylcholine (PC) biosynthesis in bone formation. Thus, we characterized PC metabolism in both primary human osteoblasts (HOB) and human osteosarcoma MG-63 cells. Our results show that the CDP-choline pathway is the only de novo route for PC biosynthesis in both HOB and MG-63 cells. Both CK activity and CKα expression in MG-63 cells were significantly higher than those in HOB cells. Silencing of CKα in MG-63 cells had no significant effect on PC concentration but decreased the amount of phosphocholine by approximately 80%. The silencing of CKα also reduced cell proliferation. Moreover, pharmacological inhibition of CK activity impaired the mineralization capacity of MG-63 cells. Our data suggest that CK and its product phosphocholine are required for the normal growth and mineralization of MG-63 cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Molecular cloning and characterization of a novel human kinase ...

    Indian Academy of Sciences (India)

    Unknown

    Molecular cloning and sequencing of PDIK1L cDNA: Human foetal brain polyA+ RNA was purchased from Clontech. (Cat. No. 6525-1). Double-stranded cDNA was prepared with the SMART PCR cDNA Synthesis kit (Clontech,. Cat. No. K1052-1) according to the manufacturer's re- commendation. The cDNAs were digested ...

  10. Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme

    DEFF Research Database (Denmark)

    Niefind, K; Guerra, B; Ermakowa, I

    2001-01-01

    The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalyt...

  11. Extracellular-regulated kinase 1/2, Jun N-terminal kinase, and c-Jun are involved in NF-kappa B-dependent IL-6 expression in human monocytes

    NARCIS (Netherlands)

    Tuyt, LML; Dokter, WHA; Birbenkamp, K; Koopmans, S.B.; Lummen, C; Kruijer, W; Vellenga, E

    1999-01-01

    In the present study we investigated the possible involvement of the mitogen-activated protein kinase family members extracellular-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in mediating IL-6 gene expression in human monocytes, in particular their role in enhancing NF-kappa B

  12. A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers.

    Science.gov (United States)

    Bischoff, J R; Anderson, L; Zhu, Y; Mossie, K; Ng, L; Souza, B; Schryver, B; Flanagan, P; Clairvoyant, F; Ginther, C; Chan, C S; Novotny, M; Slamon, D J; Plowman, G D

    1998-06-01

    Genetic and biochemical studies in lower eukaryotes have identified several proteins that ensure accurate segregation of chromosomes. These include the Drosophila aurora and yeast Ipl1 kinases that are required for centrosome maturation and chromosome segregation. We have identified two human homologues of these genes, termed aurora1 and aurora2, that encode cell-cycle-regulated serine/threonine kinases. Here we demonstrate that the aurora2 gene maps to chromosome 20q13, a region amplified in a variety of human cancers, including a significant number of colorectal malignancies. We propose that aurora2 may be a target of this amplicon since its DNA is amplified and its RNA overexpressed, in more than 50% of primary colorectal cancers. Furthermore, overexpression of aurora2 transforms rodent fibroblasts. These observations implicate aurora2 as a potential oncogene in many colon, breast and other solid tumors, and identify centrosome-associated proteins as novel targets for cancer therapy.

  13. Human cytosolic thymidine kinase: purification and physical characterization of the enzyme from HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Sherley, J.L.; Kelly, T.J.

    1988-01-05

    The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, the authors have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic R/sub m/ of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of M/sub r/ = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity.

  14. RO0504985 is an inhibitor of CMGC kinase proteins and has anti-human cytomegalovirus activity.

    Science.gov (United States)

    Strang, Blair L

    2017-08-01

    Public-private partnerships allow many previously unavailable compounds to be screened for antiviral activity. Here a screening method was used to identify an oxindole compound, RO0504985, from a Roche kinase inhibitor library that inhibited human cytomegalovirus (HCMV) protein production. RO0504985 was previously described as an inhibitor of cyclin-dependent kinase 2 (CDK2). However, using kinase selectivity assays it was found that RO0504985 was an inhibitor of several CMGC group kinase proteins, including CDK2. Using virus yield reduction assays it was observed that RO0504985 inhibited replication of different HCMV strains at low micromolar concentrations. Western blotting was used to investigate how RO0504985 inhibited HCMV replication. Treatment of HCMV infected cells with RO0504985 inhibited production of the immediate early viral IE2 proteins and the late viral protein pp28. Thus, RO0504985 inhibited HCMV replication by preventing production of specific HCMV proteins necessary for virus replication. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  15. [Identification of a novel human MAST4 gene, a new member of the microtubule associated serine-threonine kinase family].

    Science.gov (United States)

    Sun, L; Gu, S; Li, X; Sun, Y; Zheng, D; Yu, K; Ji, C; Tang, R; Xie, Y; Mao, Y

    2006-01-01

    Human protein kinases make up a large superfamily of homologous proteins, which are related by virtue of their kinase domains (also known as catalytic domains). Here we report the cloning and characterization of a novel human MAST4 (microtubule associated serine/threonine kinase family member 4) gene, which locates on human chromosome 5q13. The MAST4 cDNA is 7587 base pairs in length and encodes a putative protein of 2435 amino acids which contains a serine/threonine kinase domain and a PDZ domain. MAST4 protein has 64%, 63%, 59% and 39% identical aminoacid residues with MAST1, MAST2, MAST3 and MASTL respectively. RT-PCR analysis revealed relatively high expression level of MAST4 in most normal human tissues, with an exception of in testis, small intestine, colon and peripheral blood leukocyte.

  16. Parasite Mitogen-Activated Protein Kinases as Drug Discovery Targets to Treat Human Protozoan Pathogens

    Directory of Open Access Journals (Sweden)

    Michael J. Brumlik

    2011-01-01

    Full Text Available Protozoan pathogens are a highly diverse group of unicellular organisms, several of which are significant human pathogens. One group of protozoan pathogens includes obligate intracellular parasites such as agents of malaria, leishmaniasis, babesiosis, and toxoplasmosis. The other group includes extracellular pathogens such as agents of giardiasis and amebiasis. An unfortunate unifying theme for most human protozoan pathogens is that highly effective treatments for them are generally lacking. We will review targeting protozoan mitogen-activated protein kinases (MAPKs as a novel drug discovery approach towards developing better therapies, focusing on Plasmodia, Leishmania, and Toxoplasma, about which the most is known.

  17. Kinase pathway dependence in primary human leukemias determined by rapid inhibitor screening

    NARCIS (Netherlands)

    J.W. Tyner (Jeffrey); W.F. Yang (Wayne); A. Bankhead III (Armand); G. Fan (Guang); L.B. Fletcher (Luke); J. Bryant (Jade); J.M. Glover (Jason); B.H. Chang (Bill); S.E. Spurgeon (Stephen); W.H. Fleming (William); T. Kovacsovics; J. Gotlib (Jason); S.T. Oh (Stephen); M.W.N. Deininger (Michael W.); C.M. Zwaan (Christian Michel); M.L. den Boer (Monique); M.M. van den Heuvel-Eibrink (Marry); T. O'Hare (Thomas); B.J. Druker (Brian); M.M. Loriaux (Marc)

    2013-01-01

    textabstractKinases are dysregulated in most cancers, but the frequency of specific kinase mutations is low, indicating a complex etiology in kinase dysregulation. Here, we report a strategy to rapidly identify functionally important kinase targets, irrespective of the etiology of kinase pathway

  18. Human ether-à-go-go gene potassium channels are regulated by EGFR tyrosine kinase.

    Science.gov (United States)

    Wu, Wei; Dong, Ming-Qing; Wu, Xing-Gang; Sun, Hai-Ying; Tse, Hung-Fat; Lau, Chu-Pak; Li, Gui-Rong

    2012-02-01

    Human ether á-go-go gene potassium channels (hEAG1 or Kv10.1) are expressed in brain and various human cancers and play a role in neuronal excitement and tumor progression. However, the functional regulation of hEAG channels by signal transduction is not fully understood. The present study was therefore designed to investigate whether hEAG1 channels are regulated by protein tyrosine kinases (PTKs) in HEK 293 cells stably expressing hEAG1 gene using whole-cell patch voltage-clamp, immunoprecipitation, Western blot, and mutagenesis approaches. We found that the selective epidermal growth factor receptor (EGFR) kinase inhibitor AG556 (10 μM), but not the platelet growth factor receptor (PDGFR) kinase inhibitor AG1295 (10 μM) or the Src-family inhibitor PP2 (10 μM), can inhibit hEAG1 current, and the inhibitory effect can be reversed by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hEAG1 channels was reduced by AG556, and the reduction was significantly countered by orthovanadate. The hEAG1 mutants Y90A, Y344A and Y485A, but not Y376A and Y479A, exhibited reduced response to AG556. Interestingly, the inhibition effect of AG556 was lost in triple mutant hEAG1 channels at Y90, Y344, and Y485 with alanine. These results demonstrate for the first time that hEAG1 channel activity is regulated by EGFR kinase at the tyrosine residues Tyr90, Try344, and Try485. This effect is likely involved in regulating neuronal activity and/or tumor growth. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Relationship Between Buprenorphine Dosing and Triglyceride Lowering and Creatinine Kinase Elevation in Felines: Possible Human Implications.

    Science.gov (United States)

    Srinivas, Nuggehally R

    2016-01-01

    Recently published feline data suggest that high doses of buprenorphine can elevate creatinine kinase (CK) and profoundly influence triglyceride levels in an inverted dose versus effect relationship. This intriguing observation in felines, hitherto not documented for buprenorphine, should be considered in human situations for any trends of translatability. The report evaluates the observed effects in domestic cats and what is known about buprenorphine in human subjects. Based on the objective assessment, the following are deduced: (a) although elevated CK levels is a nonissue in humans, one needs to pay attention especially when buprenorphine is used at the high end of therapeutic dose range in the presence of drugs that can impair the hepatic metabolism of buprenorphine; and (b) the potential for triglyceride lowering can be easily confirmed in human trials, and since it may occur at the relevant therapeutic doses of buprenorphine, it may be beneficial in such patients who may have added cardiovascular risk factors.

  20. Inositol trisphosphate 3-kinase B is increased in human Alzheimer brain and exacerbates mouse Alzheimer pathology.

    Science.gov (United States)

    Stygelbout, Virginie; Leroy, Karelle; Pouillon, Valérie; Ando, Kunie; D'Amico, Eva; Jia, Yonghui; Luo, H Robert; Duyckaerts, Charles; Erneux, Christophe; Schurmans, Stéphane; Brion, Jean-Pierre

    2014-02-01

    ITPKB phosphorylates inositol 1,4,5-trisphosphate into inositol 1,3,4,5-tetrakisphosphate and controls signal transduction in various hematopoietic cells. Surprisingly, it has been reported that the ITPKB messenger RNA level is significantly increased in the cerebral cortex of patients with Alzheimer's disease, compared with control subjects. As extracellular signal-regulated kinases 1/2 activation is increased in the Alzheimer brain and as ITPKB is a regulator of extracellular signal-regulated kinases 1/2 activation in some hematopoietic cells, we tested whether this increased activation in Alzheimer's disease might be related to an increased activity of ITPKB. We show here that ITPKB protein level was increased 3-fold in the cerebral cortex of most patients with Alzheimer's disease compared with control subjects, and accumulated in dystrophic neurites associated to amyloid plaques. In mouse Neuro-2a neuroblastoma cells, Itpkb overexpression was associated with increased cell apoptosis and increased β-secretase 1 activity leading to overproduction of amyloid-β peptides. In this cellular model, an inhibitor of mitogen-activated kinase kinases 1/2 completely prevented overproduction of amyloid-β peptides. Transgenic overexpression of ITPKB in mouse forebrain neurons was not sufficient to induce amyloid plaque formation or tau hyperphosphorylation. However, in the 5X familial Alzheimer's disease mouse model, neuronal ITPKB overexpression significantly increased extracellular signal-regulated kinases 1/2 activation and β-secretase 1 activity, resulting in exacerbated Alzheimer's disease pathology as shown by increased astrogliosis, amyloid-β40 peptide production and tau hyperphosphorylation. No impact on pathology was observed in the 5X familial Alzheimer's disease mouse model when a catalytically inactive ITPKB protein was overexpressed. Together, our results point to the ITPKB/inositol 1,3,4,5-tetrakisphosphate/extracellular signal-regulated kinases 1

  1. Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway.

    Science.gov (United States)

    Chuang, Wen-Ying; Kung, Po-Hsiung; Kuo, Chih-Yun; Wu, Chin-Chung

    2013-06-01

    Sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A2 mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y12 receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.

  2. Structure of the human autophagy initiating kinase ULK1 in complex with potent inhibitors.

    Science.gov (United States)

    Lazarus, Michael B; Novotny, Chris J; Shokat, Kevan M

    2015-01-16

    Autophagy is a conserved cellular process that involves the degradation of cellular components for energy maintenance and cytoplasmic quality control that has recently gained interest as a novel target for a variety of human diseases, including cancer. A prime candidate to determine the potential therapeutic benefit of targeting autophagy is the kinase ULK1, whose activation initiates autophagy. Here, we report the first structures of ULK1, in complex with multiple potent inhibitors. These structures show features unique to the enzyme and will provide a path for the rational design of selective compounds as cellular probes and potential therapeutics.

  3. Insights into the Phosphoryl Transfer Mechanism of Human Ubiquitous Mitochondrial Creatine Kinase.

    Science.gov (United States)

    Li, Quanjie; Fan, Shuai; Li, Xiaoyu; Jin, Yuanyuan; He, Weiqing; Zhou, Jinming; Cen, Shan; Yang, ZhaoYong

    2016-12-02

    Human ubiquitous mitochondrial creatine kinase (uMtCK) is responsible for the regulation of cellular energy metabolism. To investigate the phosphoryl-transfer mechanism catalyzed by human uMtCK, in this work, molecular dynamic simulations of uMtCK∙ATP-Mg2+∙creatine complex and quantum mechanism calculations were performed to make clear the puzzle. The theoretical studies hereof revealed that human uMtCK utilizes a two-step dissociative mechanism, in which the E227 residue of uMtCK acts as the catalytic base to accept the creatine guanidinium proton. This catalytic role of E227 was further confirmed by our assay on the phosphatase activity. Moreover, the roles of active site residues in phosphoryl transfer reaction were also identified by site directed mutagenesis. This study reveals the structural basis of biochemical activity of uMtCK and gets insights into its phosphoryl transfer mechanism.

  4. Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit

    DEFF Research Database (Denmark)

    Ermakova, Inessa; Boldyreff, Brigitte; Issinger, Olaf-Georg

    2003-01-01

    Protein kinase CK2 (formerly called: casein kinase 2) is a heterotetrameric enzyme composed of two separate catalytic chains (CK2alpha) and a stable dimer of two non-catalytic subunits (CK2beta). CK2alpha is a highly conserved member of the superfamily of eukaryotic protein kinases. The crystal...... main regions of conformational plasticity and regulatory importance in eukaryotic protein kinases, in active conformations stabilized by extensive contacts to the N-terminal segment. This arrangement is in accordance with the constitutive activity of the enzyme. By structural superimposition of human...... CK2alpha in isolated form and embedded in the human CK2 holoenzyme the loop connecting the strands beta4 and beta5 and the ATP-binding loop were identified as elements of structural variability. This structural comparison suggests that the ATP-binding loop may be the key region by which the non...

  5. Crystal structure of human AKT1 with an allosteric inhibitor reveals a new mode of kinase inhibition.

    Directory of Open Access Journals (Sweden)

    Wen-I Wu

    Full Text Available AKT1 (NP_005154.2 is a member of the serine/threonine AGC protein kinase family involved in cellular metabolism, growth, proliferation and survival. The three human AKT isozymes are highly homologous multi-domain proteins with both overlapping and distinct cellular functions. Dysregulation of the AKT pathway has been identified in multiple human cancers. Several clinical trials are in progress to test the efficacy of AKT pathway inhibitors in treating cancer. Recently, a series of AKT isozyme-selective allosteric inhibitors have been reported. They require the presence of both the pleckstrin-homology (PH and kinase domains of AKT, but their binding mode has not yet been elucidated. We present here a 2.7 Å resolution co-crystal structure of human AKT1 containing both the PH and kinase domains with a selective allosteric inhibitor bound in the interface. The structure reveals the interactions between the PH and kinase domains, as well as the critical amino residues that mediate binding of the inhibitor to AKT1. Our work also reveals an intricate balance in the enzymatic regulation of AKT, where the PH domain appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain. This information advances our knowledge in AKT1 structure and regulation, thereby providing a structural foundation for interpreting the effects of different classes of AKT inhibitors and designing selective ones.

  6. Comparative Analysis of Human Nucleoside Kinase-Based Reporter Systems for PET Imaging.

    Science.gov (United States)

    Lee, Jason T; Zhang, Hanwen; Moroz, Maxim A; Likar, Yury; Shenker, Larissa; Sumzin, Nikita; Lobo, Jose; Zurita, Juan; Collins, Jeffrey; van Dam, R Michael; Ponomarev, Vladimir

    2017-02-01

    Radionuclide-based reporter gene imaging has the sensitivity to monitor gene- and cell-based therapies in human subjects. Potential immunogenicity of current viral transgenes warrants development of human-based reporter systems. We compared human nucleoside kinase reporters to a panel of nucleoside analogs of FEAU, FMAU, and FIAU, including the first in vivo assessment of L-[18F]FEAU. Human isogenic U87 cell lines were transduced to express different human reporter genes including dCK-R104M/D133A (dCKDM), dCK-R104Q/D133N (dCKep16A), dCK-A100V/R104M/D133A (dCK3M), and TK2-N93D/L109F (TK2DM), and wild-type dCK (dCK) and herpes simplex virus type-1 (HSVTK) reporter gene as references. In vitro cell uptake assays were performed with [18F]FEAU, L-[18F]FEAU, [14C]FMAU, L-[18F]FMAU, and [124I]FIAU. Micro-positron emission tomography/X-ray computed tomography imaging of xenograft-bearing nu/nu mice was conducted with [18F]FEAU, L-[18F]FEAU, L-[18F]FMAU, and [124I]FIAU on consecutive days. A cell viability assay was also performed to assess sensitivities to gemcitabine and bromovinyldeoxyuridine (BVdU). In vitro, dCKep16A and dCKDM with [18F]FEAU exhibited the highest sensitivity and selectivity of the human reporters, second only to HSVTK/[18F]FEAU. L-[18F]FEAU biodistribution in mice was on par with [18F]FEAU and L-[18F]FMAU. L-[18F]FMAU uptake in isogenic xenografts was highest for all human reporter genes. However, [18F]FEAU was the most selective of the short half-life reporter probes due to its minimal recognition by human dCK and relative sensitivity, whereas [124I]FIAU permitted imaging at a later time point, improving signal-to-background ratio. Of the human reporter genes, dCKep16A consistently outperformed the other tested reporters. Reporter genes of interest increased potency to the nucleoside analog prodrugs gemcitabine and BVdU. We demonstrate that human nucleoside kinase reporter systems vary significantly in their sensitivity and selectivity for in vivo

  7. Insights into the binding mode of MEK type-III inhibitors. A step towards discovering and designing allosteric kinase inhibitors across the human kinome

    OpenAIRE

    Zhao, Zheng; Xie, Lei; Bourne, Philip E.

    2017-01-01

    Protein kinases are critical drug targets for treating a large variety of human diseases. Type-III kinase inhibitors have attracted increasing attention as highly selective therapeutics. Thus, understanding the binding mechanism of existing type-III kinase inhibitors provides useful insights into designing new type-III kinase inhibitors. In this work, we have systematically studied the binding mode of MEK-targeted type-III inhibitors using structural systems pharmacology and molecular dynamic...

  8. Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1.

    Directory of Open Access Journals (Sweden)

    Zhen Dou

    Full Text Available BACKGROUND: Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC, a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1 is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known. METHODOLOGY/PRINCIPAL FINDINGS: Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus. CONCLUSIONS/SIGNIFICANCE: hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.

  9. Dissecting the role of AMP-activated protein kinase in human diseases

    Directory of Open Access Journals (Sweden)

    Jin Li

    2017-05-01

    Full Text Available AMP-activated protein kinase (AMPK, known as a sensor and a master of cellular energy balance, integrates various regulatory signals including anabolic and catabolic metabolic processes. Accompanying the application of genetic methods and a plethora of AMPK agonists, rapid progress has identified AMPK as an attractive therapeutic target for several human diseases, such as cancer, type 2 diabetes, atherosclerosis, myocardial ischemia/reperfusion injury and neurodegenerative disease. The role of AMPK in metabolic and energetic modulation both at the intracellular and whole body levels has been reviewed elsewhere. In the present review, we summarize and update the paradoxical role of AMPK implicated in the diseases mentioned above and put forward the challenge encountered. Thus it will be expected to provide important clues for exploring rational methods of intervention in human diseases.

  10. Effects of 5-aza-2′deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, X.Q. [Department of Oral and Maxillofacial Surgery, School of Stomatology, Shandong University, Jinan (China); Department of Oral and Maxillofacial Surgery, The First People' s Hospital of Jining, Shandong (China); Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital, Affiliated to Shandong University, Jinan (China); Huang, S.Y. [Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital, Affiliated to Shandong University, Jinan (China); Zhang, D.S. [Department of Oral and Maxillofacial Surgery, School of Stomatology, Shandong University, Jinan (China); Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital, Affiliated to Shandong University, Jinan (China); Zhang, S.Z.; Li, W.G.; Chen, Z.W.; Wu, H.W. [Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital, Affiliated to Shandong University, Jinan (China)

    2014-12-12

    Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoid cystic carcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza-2′deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation-specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECK gene hypermethylation, which might be a promising chemotherapy approach in SACC treatment.

  11. Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5' AMP-activated protein kinase activation.

    Science.gov (United States)

    Shabani Nashtaei, M; Amidi, F; Sedighi Gilani, M A; Aleyasin, A; Bakhshalizadeh, Sh; Naji, M; Nekoonam, S

    2017-03-01

    Biochemical and physical modifications during the freeze-thaw process adversely influence the restoration of energy-dependent sperm functions required for fertilization. Resveratrol, a phytoalexin, has been introduced to activate 5' AMP-activated protein kinase which is a cell energy sensor and a cell metabolism regulator. The cryoprotection of resveratrol on sperm cryoinjury via activation of AMP-activated protein kinase also remains to be elucidated. Our aim, thus, was to investigate: (i) the presence and intracellular localization of AMP-activated protein kinase protein; (ii) whether resveratrol may exert a protective effect on certain functional properties of fresh and post-thaw human spermatozoa through modulation of AMP-activated protein kinase. Spermatozoa from normozoospermic men were incubated with or without different concentrations of Compound C as an AMP-activated protein kinase inhibitor or resveratrol as an AMP-activated protein kinase activator for different lengths of time and were then cryopreserved. AMP-activated protein kinase is expressed essentially in the entire flagellum and the post-equatorial region. Viability of fresh spermatozoa was not significantly affected by the presence of Compound C or resveratrol. However, although Compound C caused a potent inhibition of spermatozoa motility parameters, resveratrol did not induce negative effect, except a significant reduction in motility at 25 μm for 1 h. Furthermore, resveratrol significantly increased AMP-activated protein kinase phosphorylation and mitochondrial membrane potential and decreased reactive oxygen species and apoptosis-like changes in frozen-thawed spermatozoa. Nevertheless, it was not able to compensate decreased sperm viability and motility parameters following cryopreservation. In contrast, Compound C showed opposite effects to resveratrol on AMP-activated protein kinase phosphorylation, reactive oxygen species, apoptosis-like changes, mitochondrial membrane potential, and

  12. Cholesterol selectively regulates IL-5 induced mitogen activated protein kinase signaling in human eosinophils.

    Directory of Open Access Journals (Sweden)

    Mandy E Burnham

    Full Text Available Eosinophils function contributes to human allergic and autoimmune diseases, many of which currently lack curative treatment. Development of more effective treatments for eosinophil-related diseases requires expanded understanding of eosinophil signaling and biology. Cell signaling requires integration of extracellular signals with intracellular responses, and is organized in part by cholesterol rich membrane microdomains (CRMMs, commonly referred to as lipid rafts. Formation of these organizational membrane domains is in turn dependent upon the amount of available cholesterol, which can fluctuate widely with a variety of disease states. We tested the hypothesis that manipulating membrane cholesterol content in primary human peripheral blood eosinophils (PBEos would selectively alter signaling pathways that depend upon membrane-anchored signaling proteins localized within CRMMs (e.g., mitogen activated protein kinase [MAPK] pathway, while not affecting pathways that signal through soluble proteins, like the Janus Kinase/Signal Transducer and Activator of Transcription [JAK/STAT] pathway. Cholesterol levels were increased or decreased utilizing cholesterol-chelating methyl-β-cyclodextrin (MβCD, which can either extract membrane cholesterol or add exogenous membrane cholesterol depending on whether MβCD is preloaded with cholesterol. Human PBEos were pretreated with MβCD (cholesterol removal or MβCD+Cholesterol (MβCD+Chol; cholesterol delivery; subsequent IL-5-stimulated signaling and physiological endpoints were assessed. MβCD reduced membrane cholesterol in PBEos, and attenuated an IL-5-stimulated p38 and extracellular-regulated kinase 1/2 phosphorylation (p-p38, p-ERK1/2, and an IL-5-dependent increase in interleukin-1β (IL-1β mRNA levels. In contrast, MβCD+Chol treatment elevated PBEos membrane cholesterol levels and basal p-p38, but did not alter IL-5-stimulated phosphorylation of ERK1/2, STAT5, or STAT3. Furthermore, M

  13. Coexpression of receptor tyrosine kinase AXL and EGFR in human primary lung adenocarcinomas.

    Science.gov (United States)

    Wu, Zhenzhou; Bai, Fan; Fan, Liyun; Pang, Wenshuai; Han, Ruiyu; Wang, Juan; Liu, Yueping; Yan, Xia; Duan, Huijun; Xing, Lingxiao

    2015-12-01

    AXL has been identified as a tyrosine kinase switch that causes resistance to inhibitors targeting epidermal growth factor receptor (EGFR) signaling in non-small cell lung cancer (NSCLC). However, the relationship between 2 receptor tyrosine kinases, AXL and EGFR, and the relevance of AXL expression with EGFR mutation status in treatment-naive human NSCLCs remain uncertain. In this study, we evaluated the coexpression pattern of AXL, EGFR, and pEGFR(1068) in 109 lung adenocarcinoma patients with or without an EGFR mutation. There were 68 (62.4%) patients with tumors harboring EGFR mutations such as 19 del and/or L858R; 2 patients were T790M positive. The expression of AXL, EGFR, and pEGFR(1068) was detected in 60 (55%), 68 (62.4%), and 57 (52.3%) of 109 patients, respectively. The positive rates of EGFR and pEGFR(1068) were associated with the L858R mutation alone or with the 19 del and L858R mutation status. Further analysis indicated that the percentage of AXL(+)/EGFR(+)/pEGFR(1068) coexpression in 68 EGFR-activating mutations patients was significantly higher than that in 39 EGFR wild-type patients (30.9% versus 10.3%, P=.015). Furthermore, in the subgroup of AXL(+) patients (35 mutation(+) and 23 wild-type patients), the coexpression rates of AXL(+)/pEGFR(1068+) and AXL(+)/EGFR(+)/pEGFR(1068+) in patients with EGFR mutations were significantly higher compared with those in wild-type patients (both P<.05). Our study emphasized that the AXL and EGFR receptor tyrosine kinases were coexpressed in a subgroup of treatment-naive lung adenocarcinomas with or without EGFR mutations. Anti-AXL therapeutics delivered up front in combination with an EGFR inhibitor might prevent or delay resistance in patients with AXL-positive, EGFR-mutant, or wild-type NSCLC. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Crystal structure of the Sema-PSI extracellular domain of human RON receptor tyrosine kinase.

    Directory of Open Access Journals (Sweden)

    Kinlin L Chao

    Full Text Available Human RON (Recepteur d'Origine Nantais receptor tyrosine kinase is a cell surface receptor for Macrophage Stimulating Protein (MSP. RON mediates signal transduction pathways that regulate cell adhesion, invasion, motility and apoptosis processes. Elevated levels of RON and its alternatively spliced variants are implicated in the progression and metastasis of tumor cells. The binding of MSP α/β heterodimer to the extracellular region of RON receptor induces receptor dimerization and activation by autophosphorylation of the intracellular kinase domains. The ectodomain of RON, containing the ligand recognition and dimerization domains, is composed of a semaphorin (Sema, Plexins-Semaphorins-Integrins domain (PSI, and four Immunoglobulins-Plexins-Transcription factor (IPT domains. High affinity association between MSP and RON is mediated by the interaction between MSP β-chain and RON Sema, although RON activation requires intact RON and MSP proteins. Here, we report the structure of RON Sema-PSI domains at 1.85 Å resolution. RON Sema domain adopts a seven-bladed β-propeller fold, followed by disulfide bond rich, cysteine-knot PSI motif. Comparison with the homologous Met receptor tyrosine kinase reveals that RON Sema-PSI contains distinguishing secondary structural features. These define the receptors' exclusive selectivity towards their respective ligands, RON for MSP and Met for HGF. The RON Sema-PSI crystal packing generates a homodimer with interface formed by the Sema domain. Mapping of the dimer interface using the RON homology to Met, MSP homology to Hepatocyte Growth Factor (HGF, and the structure of the Met/HGF complex shows the dimer interface overlapping with the putative MSPβ binding site. The crystallographically determined RON Sema-PSI homodimer may represent the dimer assembly that occurs during ligand-independent receptor activation and/or the inhibition of the constitutive activity of RONΔ160 splice variant by the soluble RON

  15. Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini

    Energy Technology Data Exchange (ETDEWEB)

    Grove, J.R.; Banerjee, P.; Balasubramanyam, A.; Price, D.J.; Avruch, J. (Harvard Medical School, Boston, MA (United States)); Coffer, P.J.; Woodgett, J.R. (Ludwig Inst. for Cancer Research, London (United Kingdom))

    1991-11-01

    Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5{prime} region, such that the longer polypeptide consists of 525 amino acids, of which the last 502 longer residues are identical in sequence to the entire polypeptide encoded by the second cDNA. Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase {alpha}I cDNA through the utilization of different translational start sites. Transient expression of p70 {alpha}I and {alpha}II S6 kinase cDNA in COA cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Transfection with the {alpha}II cDNA yields only the smaller set of bands, while transfection with the {alpha}I cDNA generates both sets of bands. Mutation of Met-24 in the {alpha}I cDNA to Leu or Thr suppresses synthesis of the {alpha}II polypeptides. Only the p70 {alpha}I and {alpha}II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70-and 90-kDa proteins observed in purified rat liver S6 kinase. The recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.

  16. Studies on adenosine triphosphate transphosphorylases. Human isoenzymes of adenylate kinase: isolation and physicochemical comparison of the crystalline human ATP-AMP transphosphorylases from muscle and liver.

    Science.gov (United States)

    Kuby, S A; Fleming, G; Frischat, A; Cress, M C; Hamada, M

    1983-02-10

    Procedures are described for the isolation, in crystalline form, of the adenylate kinases from autopsy samples of human muscle and from human liver. Weight average molecular weights were determined by sedimentation equilibrium to be 22,000 (+/- 700) and 25,450 (+/- 160) for the human muscle and liver isoenzymes, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their molecular weights were estimated to be 21,700 and 26,500 for the muscle and liver enzymes, respectively. Both isoenzymes are accordingly monomeric proteins in their native state. Amino acid analyses are reported here for the normal human liver, calf liver, and rabbit liver adenylate kinases and compared with the normal human muscle, calf muscle, and rabbit muscle myokinases. The liver types as a group and the muscle types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, half-cystine, and the presence of tryptophan in the liver enzymes. The normal human liver adenylate kinase, as isolated in this report, has proved to be similar in its properties, if not identical, to the adenylate kinase isolated directly from human liver mitochondria (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S. A. (1982) J. Biol. Chem. 257, 13120-13128). Therefore, the liver-type adenylate kinase may be considered a mitochondrial type.

  17. Sanguinarine causes cell cycle blockade and apoptosis of human prostate carcinoma cells via modulation of cyclin kinase inhibitor-cyclin-cyclin-dependent kinase machinery.

    Science.gov (United States)

    Adhami, Vaqar Mustafa; Aziz, Moammir Hasan; Reagan-Shaw, Shannon R; Nihal, Minakshi; Mukhtar, Hasan; Ahmad, Nihal

    2004-08-01

    Prostate cancer is the second leading cause of cancer-related deaths in males in the United States. This warrants the development of novel mechanism-based strategies for the prevention and/or treatment of prostate cancer. Several studies have shown that plant-derived alkaloids possess remarkable anticancer effects. Sanguinarine, an alkaloid derived from the bloodroot plant Sanguinaria canadensis, has been shown to possess antimicrobial, anti-inflammatory, and antioxidant properties. Previously, we have shown that sanguinarine possesses strong antiproliferative and proapoptotic properties against human epidermoid carcinoma A431 cells and immortalized human HaCaT keratinocytes. Here, employing androgen-responsive human prostate carcinoma LNCaP cells and androgen-unresponsive human prostate carcinoma DU145 cells, we studied the antiproliferative properties of sanguinarine against prostate cancer. Sanguinarine (0.1-2 micromol/L) treatment of LNCaP and DU145 cells for 24 hours resulted in dose-dependent (1) inhibition of cell growth [as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], (2) arrest of cells in G0-G1 phase of the cell cycle (as assessed by DNA cell cycle analysis), and (3) induction of apoptosis (as evaluated by DNA ladder formation and flow cytometry). To define the mechanism of antiproliferative effects of sanguinarine against prostate cancer, we studied the effect of sanguinarine on critical molecular events known to regulate the cell cycle and the apoptotic machinery. Immunoblot analysis showed that sanguinarine treatment of both LNCaP and DU145 cells resulted in significant (1) induction of cyclin kinase inhibitors p21/WAF1 and p27/KIP1; (2) down-regulation of cyclin E, D1, and D2; and (3) down-regulation of cyclin-dependent kinase 2, 4, and 6. A highlight of this study was the fact that sanguinarine induced growth inhibitory and antiproliferative effects in human prostate carcinoma cells irrespective of their androgen

  18. Functional Analysis of the Aurora Kinase A Ile31 Allelic Variant in Human Prostate

    Directory of Open Access Journals (Sweden)

    Noa Matarasso

    2007-09-01

    Full Text Available Overexpression of the centrosome-associated serine/ threonine kinase Aurora Kinase A (AURKA has been demonstrated in both advanced prostate cancer and high-grade prostatic intraepithelial neoplasia lesions. The single-nucleotide polymorphism T91A (Phe3lile has been implicated in AURKA overexpression and has been suggested as a low-penetrance susceptibility allele in multiple human cancers, including prostate cancer. We studied the transcriptional consequences of the AURKA Ile31 allele in 28 commercial normal prostate tissue RNA samples (median age, 27 years. Significant overexpression of AURKA was demonstrated in homozygous and heterozygous AURKA Ile31 prostate RNA (2.07-fold and 1.93-fold, respectively; P < .05. Expression levels of 1509 genes differentiated between samples homozygous for Phe31 alleles and samples homozygous for Ile31 alleles (P = .05. Gene Ontology classification revealed overrepresentation of cell cycle arrest, ubiquitin cycle, antiapoptosis, angiogenesisrelated genes. When these hypothesis-generating results were subjected to more stringent statistical criteria, overexpression of a novel transcript of the natural killer tumor recognition sequence (NKTR gene was revealed and validated in homozygous Ile31 samples (2.6-fold; P < .05. In summary, our data suggest an association between the AURKA Ile31 allele and an altered transcriptome in normal non-neopasic prostates.

  19. Integrin-Associated Focal Adhesion Kinase Protects Human Embryonic Stem Cells from Apoptosis, Detachment, and Differentiation

    Directory of Open Access Journals (Sweden)

    Loriana Vitillo

    2016-08-01

    Full Text Available Human embryonic stem cells (hESCs can be maintained in a fully defined niche on extracellular matrix substrates, to which they attach through integrin receptors. However, the underlying integrin signaling mechanisms, and their contribution to hESC behavior, are largely unknown. Here, we show that focal adhesion kinase (FAK transduces integrin activation and supports hESC survival, substrate adhesion, and maintenance of the undifferentiated state. After inhibiting FAK kinase activity we show that hESCs undergo cell detachment-dependent apoptosis or differentiation. We also report deactivation of FAK downstream targets, AKT and MDM2, and upregulation of p53, all key players in hESC regulatory networks. Loss of integrin activity or FAK also induces cell aggregation, revealing a role in the cell-cell interactions of hESCs. This study provides insight into the integrin signaling cascade activated in hESCs and reveals in FAK a key player in the maintenance of hESC survival and undifferentiated state.

  20. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

  1. The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins

    Directory of Open Access Journals (Sweden)

    Laura Graf

    2013-12-01

    Full Text Available The human cytomegalovirus (HCMV-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231–280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins.

  2. The Human Biliverdin Reductase-based Peptide Fragments and Biliverdin Regulate Protein Kinase Cδ Activity

    Science.gov (United States)

    Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E. M.; Tudor, Cicerone; Hagen, Fred K.; Maines, Mahin D.

    2012-01-01

    PKCδ, a Ser/Thr kinase, promotes cell growth, tumorigenesis, and apoptosis. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase, inhibits apoptosis by reducing biliverdin-IX to antioxidant bilirubin. The enzymes are activated by similar stimuli. Reportedly, hBVR is a kinase-independent activator of PKCδ and is transactivated by the PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C., and Maines, M. D. (2012) J. Biol. Chem. 287, 1066–1079). Presently, we examined interactions between the two proteins in the context of regulation of their activities and defining targets of hBVR phosphorylation by PKCδ. LC-MS/MS analysis of PKCδ-activated intact hBVR identified phosphorylated serine positions 21, 33, 230, and 237, corresponding to the hBVR Src homology-2 domain motif (Ser230 and Ser237), flanking the ATP-binding motif (Ser21) and in PHPS sequence (Ser33) as targets of PKCδ. Ser21 and Ser230 were also phosphorylated in hBVR-based peptides. The Ser230-containing peptide was a high affinity substrate for PKCδ in vitro and in cells; the relative affinity was PKCδ > PKCβII > PKCζ. Two overlapping peptides spanning this substrate, KRNRYLSF and SFHFKSGSL, were effective inhibitors of PKCδ kinase activity and PKCδ-supported activation of transcription factors Elk1 and NF-κB. Only SFHFKSGSL, in PKCδ-transfected phorbol 12-myristate 13-acetate-stimulated cells, caused membrane blebbing and cell loss. Biliverdin noncovalently inhibited PKCδ, whereas PKCδ potentiated hBVR reductase activity and accelerated the rate of bilirubin formation. This study, together with previous findings, reveals an unexpected regulatory interplay between PKCδ and hBVR in modulating cell death/survival in response to various activating stimuli. In addition, this study has identified novel substrates for and inhibitors of PKCδ. We suggest that hBVR-based technology may have utility to modulate PKCδ-mediated functions in the cell. PMID:22584576

  3. Comparative active‐site mutation study of human and Caenorhabditis elegans thymidine kinase 1

    National Research Council Canada - National Science Library

    Skovgaard, Tine; Uhlin, Ulla; Munch‐Petersen, Birgitte

    2012-01-01

    The first step for the intracellular retention of several anticancer or antiviral nucleoside analogues is the addition of a phosphate group catalysed by a deoxyribonucleoside kinase such as thymidine kinase 1 (TK1...

  4. Regulation of Cell Survival in Human Breast Cancer Cells by Sphingosine Kinase

    National Research Council Canada - National Science Library

    Hobson, John

    2001-01-01

    .... Cell migration toward PDGF, which stimulates sphingosine kinase and increases SPP levels, was dependent on expression of EDG-1, and conversely, deletion of EDG-1, inhibition of sphingosine kinase...

  5. Metformin action in human hepatocytes: coactivation of atypical protein kinase C alters 5'-AMP-activated protein kinase effects on lipogenic and gluconeogenic enzyme expression.

    Science.gov (United States)

    Sajan, Mini P; Ivey, Robert A; Farese, Robert V

    2013-11-01

    Atypical protein kinase C (aPKC) levels and activity are elevated in hepatocytes of individuals with type 2 diabetes and cause excessive increases in the levels of lipogenic and gluconeogenic enzymes; aPKC inhibitors largely correct these aberrations. Metformin improves hepatic gluconeogenesis by activating 5'-AMP-activated protein kinase (AMPK). However, metformin also activates aPKC in certain tissues; in the liver, this activation could amplify diabetic aberrations and offset the positive effects of AMPK. In this study, we examined whether metformin activates aPKC in human hepatocytes and the metabolic consequences of any such activation. We compared protein kinase activities and alterations in lipogenic and gluconeogenic enzyme levels during activity of the AMPK activators metformin and AICAR, relative to those of an aPKC-ι inhibitor, in hepatocytes from non-diabetic and type 2 diabetic human organ donors. Metformin and 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) activated aPKC at concentrations comparable with those required for AMPK activation. Moreover, both agents increased lipogenic enzyme levels by an aPKC-dependent mechanism. Thus, whereas insulin- and diabetes-dependent increases in lipogenic enzyme levels were reversed by aPKC inhibition, such levels were increased in hepatocytes from non-diabetic donors and remained elevated in hepatocytes from diabetic donors following metformin and AICAR treatment. In addition, whereas aPKC inhibition diminished gluconeogenic enzyme levels in the absence and presence of insulin in hepatocytes from both non-diabetic and diabetic donors, metformin and AICAR increased gluconeogenic enzyme levels in hepatocytes from non-diabetic individuals, but nevertheless diminished gluconeogenic enzyme levels in insulin-treated hepatocytes from diabetic donors. Metformin and AICAR activate aPKC together with AMPK in human hepatocytes. Activation of aPKC increases lipogenic enzyme levels and alters gluconeogenic

  6. Studies of the cytosolic thymidine kinase in human cells and comparison to the recombinantly expressed enzyme

    DEFF Research Database (Denmark)

    Kock Jensen, Helle

    Thymidine kinase (TK) is a key enzyme in the salvage pathway of the nucleoside metabolism catalyzing the first phosphorylation step in TTP synthesis. Human cytosolic TK (TKl) is highly cell cycle regulated. TKl is regulated on many different levels of expression and isoforms with altered enzymatic...... properties are found in cancer cells. Investigation of these factors offers possibilities to understand the molecular background for TKl expression including to clarify general regulation patterns. It also gives valuable information for constructing new nucleoside analogs for the therapy of cancer and virus...... infections. In the first part of the present investigation a sensitive test for quantitating TKl mRNA (competitive PCR) is developed and the results show that PHA stimulated lymphocytes reveal the same pattern concerning expression of TKl mRNA and TKl enzyme activity as serum-stimulated cells. This pattern...

  7. A pseudoreceptor modelling study of the varicella-zoster virus and human thymidine kinase binding sites

    Science.gov (United States)

    Greenidge, Paulette A.; Merz, Alfred; Folkers, Gerd

    1995-12-01

    A representative range of pyrimidine nucleoside analogues that are known to inhibit herpes simplex virus (HSV) replication have been used to construct receptor binding site models for the varicella-zoster virus (VZV), thymidine kinase (TK) and human TK1. Given a set of interacting ligands, superimposed in such a manner as to define a pharmacophore, the pseudoreceptor modelling technique Yak provides a means of building binding site models of macromolecules for which no three-dimensional experimental structures are available. Once the models have been evaluated by their ability to reproduce experimental binding data [Vedani et al., J. Am. Chem. Soc., 117 (1995) 4987], they can be used for predictive purposes. Calculated and experimental values of relative binding affinity are compared. Our models suggest that the substitution of one residue may be sufficient to determine ligand subtype affinity.

  8. Regulation of ERK-mediated signal transduction by p38 MAP kinase in human monocytic THP-1 cells.

    Science.gov (United States)

    Numazawa, Satoshi; Watabe, Masahiko; Nishimura, Satoshi; Kurosawa, Masahiro; Izuno, Makoto; Yoshida, Takemi

    2003-05-01

    SB 203580 has been widely used to specifically shut down the p38 MAP kinase-dependent pathway, although it is capable of inducing c-Raf kinase activity in cells. The present study demonstrates that SB 203580 activates members of the ERK cascade, c-Raf, MEK, and ERK, in human monocytic THP-1 cells. The activation of these kinases was sustained for at least 24 h after SB 203580 treatment and was also observed in U937 cells, suggesting that c-Raf efficiently transduces the signal even in the presence of the inhibitor in these cells. However, the expression of ERK cascade-dependent genes, such as c-fos and IL-1beta, was extremely limited. Analysis of the cellular distribution of ERK in SB 203580-treated cells indicated that nuclear translocation of phosphorylated ERK was impaired. Also, nuclear translocation of ERK induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was inhibited by SB 239063, which does not associate with c-Raf and is highly selective for p38 MAP kinase. In addition, the forced expression of the dominant negative mutant of p38 MAP kinase suppressed serum responsive element-dependent transactivation induced by TPA. These results suggest that the steady-state level of p38 MAP kinase activity modulates ERK signaling.

  9. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    Science.gov (United States)

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

    2009-01-01

    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

  10. Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4

    NARCIS (Netherlands)

    Klaus-Heisen, D.; Nurisso, A.; Pietraszewska-Bogiel, A.; Mbengue, M.; Camut, S.; Timmers, T.; Pichereaux, C.; Rossignol, M.; Gadella, T.W.J.; Imberty, A.; Lefebvre, B.; Cullimore, J.V.

    2011-01-01

    Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like

  11. KinMutRF: a random forest classifier of sequence variants in the human protein kinase superfamily

    DEFF Research Database (Denmark)

    Pons, Tirso; Vazquez, Miguel; Matey-Hernandez, María Luisa

    2016-01-01

    remains challenging: cells tolerate most genomic alterations and only a minor fraction disrupt molecular function sufficiently and drive disease. Results: KinMutRF is a novel random-forest method to automatically identify pathogenic variants in human kinases. Twenty six decision trees implemented......Background: The association between aberrant signal processing by protein kinases and human diseases such as cancer was established long time ago. However, understanding the link between sequence variants in the protein kinase superfamily and the mechanistic complex traits at the molecular level...... as a random forest ponder a battery of features that characterize the variants: a) at the gene level, including membership to a Kinbase group and Gene Ontology terms; b) at the PFAM domain level; and c) at the residue level, the types of amino acids involved, changes in biochemical properties, functional...

  12. Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication.

    Science.gov (United States)

    Guedán, Anabel; Swieboda, Dawid; Charles, Mark; Toussaint, Marie; Johnston, Sebastian L; Asfor, Amin; Panjwani, Anusha; Tuthill, Tobias J; Danahay, Henry; Raynham, Tony; Mousnier, Aurelie; Solari, Roberto

    2017-05-01

    Picornavirus replication is known to cause extensive remodeling of Golgi and endoplasmic reticulum membranes, and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi membrane vesicular and lipid transport, we hypothesized that PKD played a role in viral replication. We present multiple lines of evidence in support of this hypothesis. First, infection of HeLa cells with human rhinovirus (HRV) induced the phosphorylation of PKD. Second, PKD inhibitors reduced HRV genome replication, protein expression, and titers in a concentration-dependent fashion and also blocked the replication of poliovirus (PV) and foot-and-mouth disease virus (FMDV) in a variety of cells. Third, HRV replication was significantly reduced in HeLa cells overexpressing wild-type and mutant forms of PKD1. Fourth, HRV genome replication was reduced in HAP1 cells in which the PKD1 gene was knocked out by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Although we have not identified the molecular mechanism through which PKD regulates viral replication, our data suggest that this is not due to enhanced interferon signaling or an inhibition of clathrin-mediated endocytosis, and PKD inhibitors do not need to be present during viral uptake. Our data show for the first time that targeting PKD with small molecules can inhibit the replication of HRV, PV, and FMDV, and therefore, PKD may represent a novel antiviral target for drug discovery.IMPORTANCE Picornaviruses remain an important family of human and animal pathogens for which we have a very limited arsenal of antiviral agents. HRV is the causative agent of the common cold, which in itself is a relatively trivial infection; however, in asthma and chronic obstructive

  13. [Cytoplasmic kinase inhibitors].

    Science.gov (United States)

    Mano, Hiroyuki

    2010-10-01

    Protein kinases play essential roles in the regulation of cell proliferation. Point mutations or/and fusions of protein kinases are frequently identified in human cancers, and targeting such activated kinases provides us with a chance to eradicate tumor cells. This was first proved by imatinib mesylate that inhibits ABL tyrosine kinase and, thereby, efficiently kills malignant cells in chronic myeloid leukemia. In addition, other clinical trials are ongoing for kinase inhibitors against EML4--ALK in lung cancer, JAK2 in myeloproliferative disorders and BRAF in malignant melanoma. Early reports indeed reveal that such targeting compounds are promising drugs for human cancers with activated kinases.

  14. Hypomethylating agent 5-aza-2'-deoxycytidine (DAC) ameliorates multiple sclerosis in mouse models

    DEFF Research Database (Denmark)

    Mangano, Katia; Fagone, Paolo; Bendtzen, Klaus

    2014-01-01

    Increasing evidence supports the role of epigenetics in the development of autoimmune disorders and the possibility of using epigenetic modifying drugs in the context of MS has not yet been investigated. We have explored the effect of the hypomethylating agent 5-aza-2'-deoxycytidine (DAC) in two...... murine models of experimental allergic encephalomyelitis (EAE). DAC treatment was associated with a significant amelioration of the clinical and histological hallmarks of EAE in both models. These effects were observed both in prophylactic and therapeutic regimens. The milder course of the disease...

  15. Synthesis of (2- sup 14 C)-2'-deoxycytidine- sup 3 N-cyanoborane

    Energy Technology Data Exchange (ETDEWEB)

    Burnham, B.S.; Wyrick, S.D.; Hall, I.H. (Univ. of North Carolina, Chapel Hill, NC (United States). School of Pharmacy); Sood, A.; Spielvogel, B.F. (Boron Biologicals Inc., Raleigh, NC (United States))

    1991-04-01

    Synthetic nucleosides (e.g., 5-fluorouracil, 6-mercaptopurine) are used extensively as antineoplastic drugs. Recent research in boron chemistry has led to the development of a series of amine-boranes that possess significant antineoplastic activity. The agents may also be useful in boron neutron capture therapy (BNCT), therefore giving the compounds a dual mechanism of cytotoxic action. Herein is a report of the synthesis of one of the more active boronated nucleosides, 2'-deoxycytidine-{sup 3}N-cyanoborane with a {sup 14}C-label in the 2-position of the pyrimidine ring. (author).

  16. SKI-606 (bosutinib), a novel Src kinase inhibitor, suppresses migration and invasion of human breast cancer cells

    Science.gov (United States)

    Vultur, Adina; Buettner, Ralf; Kowolik, Claudia; Liang, Wei; Smith, David; Boschelli, Frank; Jove, Richard

    2009-01-01

    Src family kinase (SFK) activity is elevated in many human tumors, including breast cancer, and is often associated with aggressive disease. We examined the effects of SKI-606 (bosutinib), a selective SFK inhibitor, on human cancer cells derived from breast cancer patients in order to assess its potential for breast cancer treatment. Our results show that SKI-606 caused a decrease in cell motility and invasion of breast cancer cell lines with an IC50 of ~250 nM, which was also the IC50 for inhibition of c-Src kinase activity in intact tumor cells. These changes were accompanied by an increase in cell-to-cell adhesion and membrane localization of beta-catenin. By contrast, cell proliferation and survival were unaffected by SKI-606 at concentrations sufficient to block cell migration and invasion. Analysis of downstream effectors of Src revealed that SKI-606 inhibits the phosphorylation of focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2) and Crk-associated substrate (p130Cas) with an IC50 similar to inhibition of c-Src kinase. Our findings indicate that SKI-606 inhibits signaling pathways involved in controlling tumor cell motility and invasion, suggesting that SKI-606 is a promising therapeutic for breast cancer. PMID:18483306

  17. Structure and function of adenylate kinase isozymes in normal humans and muscular dystrophy patients.

    Science.gov (United States)

    Hamada, M; Takenaka, H; Fukumoto, K; Fukamachi, S; Yamaguchi, T; Sumida, M; Shiosaka, T; Kurokawa, Y; Okuda, H; Kuby, S A

    1987-01-01

    Two isozymes of adenylate kinase from human Duchenne muscular dystrophy serum, one of which was an aberrant form specific to DMD patients, were separated by Blue Sepharose CL-6B affinity chromatography. The separated aberrant form possessed a molecular weight of 98,000 +/- 1,500, whereas the normal serum isozyme had a weight of 87,000 +/- 1,600, as determined by SDS-polyacrylamide gel electrophoresis, gel filtration, and sedimentation equilibrium. The sedimentation coefficients were 5.8 S and 5.6 S for the aberrant form and the normal form, respectively. Both serum isozymes are tetramers. The subunit size of the aberrant isozyme (Mr = 24,700) was very similar to that of the normal human liver isozyme, and the subunit size of the normal isozyme (Mr = 21,700) was very similar to that of the normal human muscle enzyme. The amino acid composition of the normal serum isozyme was similar to that of the muscle-type enzyme, and that of the aberrant isozyme was similar to that of the liver enzyme, with some exceptions in both cases.

  18. Activation of focal adhesion kinase enhances the adhesion of Fusarium solani to human corneal epithelial cells via the tyrosine-specific protein kinase signaling pathway.

    Science.gov (United States)

    Pan, Xiaojing; Wang, Ye; Zhou, Qingjun; Chen, Peng; Xu, Yuanyuan; Chen, Hao; Xie, Lixin

    2011-03-05

    To determine the role of the integrin-FAK signaling pathway triggered by the adherence of F. solani to human corneal epithelial cells (HCECs). After pretreatment with/without genistein, HCECs were incubated with F. solani spores at different times (0-24 h). Cell adhesion assays were performed by optical microscopy. Changes of the ultrastructure were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The expression of F-actin and Paxillin (PAX) were detected by immunofluorescence and western blotting to detect the expression of these key proteins with/without genistein treatment. Cell adhesion assays showed that the number of adhered spores began to rise at 6 h after incubation and peaked at 8 h. SEM and TEM showed that the HCECs exhibited a marked morphological alteration induced by the attachment and entry of the spores. The expression of PAX increased, while the expression of F-actin decreased by stimulation with F. solani. The interaction of F. solani with HCECs causes actin rearrangement in HCECs. Genistein strongly inhibited FAK phosphorylation and the activation of the downstream protein (PAX). F. solani-induced enhancement of cell adhesion ability was inhibited along with the inhibition of FAK phosphorylation. Our results suggest that the integrin-FAK signaling pathway is involved in the control of F. solani adhesion to HCECs and that the activation of focal adhesion kinase enhances the adhesion of human corneal epithelial cells to F. solani via the tyrosine-specific protein kinase signaling pathway.

  19. β3-Adrenoceptor-mediated relaxation of rat and human urinary bladder : roles of BKCa channels and Rho kinase

    NARCIS (Netherlands)

    Cernecka, Hana; Kersten, Kim; Maarsingh, Harm; Elzinga, Carolina R.; de Jong, Igle Jan; Korstanje, Cees; Michel, Martin C.; Schmidt, Martina

    Previous studies suggest that the large-conductance Ca2+-activated K+ (BKCa) channel and Rho-kinase play major roles in the control of urinary bladder tone. Here, we investigated their involvement in beta-adrenoceptor (AR)-mediated relaxation of rat and human bladder. Concentration-response curves

  20. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    Science.gov (United States)

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2* Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue11The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  1. Isoflurane Protects Against Human Endothelial Cell Apoptosis by Inducing Sphingosine Kinase-1 via ERK MAPK

    Directory of Open Access Journals (Sweden)

    H. Thomas Lee

    2012-01-01

    Full Text Available Endothelial dysfunction is a major clinical problem affecting virtually every patient requiring critical care. Volatile anesthetics are frequently used during the perioperative period and protect the heart and kidney against ischemia and reperfusion injury. We aimed to determine whether isoflurane, the most commonly used volatile anesthetic in the USA, protects against endothelial apoptosis and necrosis and the mechanisms involved in this protection. Human endothelial EA.hy926 cells were pretreated with isoflurane or carrier gas (95% room air + 5% CO2 then subjected to apoptosis with tumor necrosis factor-α or to necrosis with hydrogen peroxide. DNA laddering and in situ Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick-End Labeling (TUNEL staining determined EA.hy926 cell apoptosis and percent LDH released determined necrosis. We also determined whether isoflurane modulates the expression and activity of sphingosine kinase-1 (SK1 and induces the phosphorylation of extracellular signal regulated kinase (ERK MAPK as both enzymes are known to protect against cell death. Isoflurane pretreatment significantly decreased apoptosis in EA.hy926 cells as evidenced by reduced TUNEL staining and DNA laddering without affecting necrosis. Mechanistically, isoflurane induces the phosphorylation of ERK MAPK and increased SK1 expression and activity in EA.hy926 cells. Finally, selective blockade of SK1 (with SKI-II or S1P1 receptor (with W146 abolished the anti-apoptotic effects of isoflurane. Taken together, we demonstrate that isoflurane, in addition to its potent analgesic and anesthetic properties, protects against endothelial apoptosis most likely via SK1 and ERK MAPK activation. Our findings have significant clinical implication for protection of endothelial cells during the perioperative period and patients requiring critical care.

  2. Synthetic genes for human muscle-type adenylate kinase in Escherichia coli.

    Science.gov (United States)

    Kim, H J; Nishikawa, S; Tanaka, T; Uesugi, S; Takenaka, H; Hamada, M; Kuby, S A

    1989-01-01

    An artificial gene coding for the human muscle-type cytosolic adenylate kinase (hAK1) was chemically synthesized and directly expressed in Escherichia coli under the control of trp promoter. The DNA duplex of 596 bp was designed and constructed from 40 oligonucleotide fragments of typically 30 nucleotides in length. Twelve unique restriction sites were fairly evenly spaced in the synthetic gene to facilitate site-specific mutagenesis at any part of this recombinant protein. The genes for mutant hAK1 (Tyr 95----Phe 95, Y95F hAK1; Arg 97----Ala 97, R97A hAK1) were constructed by cassette mutagenesis and utilized restriction sites incorporated in the hAK1 gene. The recombinant hAK1 was purified to homogeneity by a two-step chromatographic procedure with a good yield, and showed the same adenylate kinase activity as that of authentic hAK1. Preliminary kinetic studies show that the enzymatic activity (Vmax app,cor/Et) of Y95F hAK1 was slightly greater than that of recombinant hAK1, whereas R97A hAK1 still possessed approximately 4% of recombinant hAK1 activity. These results suggest that the Arg-97 residue is important but not essential for catalytic activity, and that Tyr-95 can be replaced by phenylalanine without substantial effects on the enzymatic activity. Moreover, preliminary estimates of the apparent kinetic parameters suggest that these residues are not required for MgATP binding, and therefore they do not appear to be part of the MgATP binding site.

  3. Thermodynamic, structural and fluorescent characteristics of DNA hairpins containing functionalized pyrrolo-2'-deoxycytidines.

    Science.gov (United States)

    Jahnz-Wechmann, Zofia; Lisowiec-Wachnicka, Jolanta; Framski, Grzegorz; Kosman, Joanna; Boryski, Jerzy; Pasternak, Anna

    2017-04-01

    Herein, we present comprehensive physicochemical and structural analysis of various DNA hairpins modified with pyrrolo-2'-deoxycytidine (Py-dC) derivatives. The introduction of modified Py-dC in most cases causes minor decrease of hairpin thermodynamic stability. The energetically unfavorable effect is more pronounced when modified residue is present within hairpin loop. Our studies indicate that thermodynamic effects induced by all Py-dC derivatives are net results of increased stacking interactions caused by larger surface of pyrrolo-2'-deoxycytidine aromatic ring and unfavorable effect implied by the presence of additional side chains. The CD spectra of all modified hairpins are similar to unmodified hairpin indicating that the presence of Py-dC derivatives does not disrupt the secondary structure of DNA. Interestingly, the presence of various side chains can increase fluorescent discrimination of paired and unpaired regions of DNA. The fluorescence observed for hairpins modified within loop is significantly quenched when Py-dC derivative is present in the stem region. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Quaternary structures of recombinant, cellular, and serum forms of Thymidine Kinase 1 from dogs and humans

    Directory of Open Access Journals (Sweden)

    Hanan Sharif

    2012-06-01

    Full Text Available Abstract Background Thymidine kinase 1 (TK1 is a salvage enzyme involved in DNA precursor synthesis, and its expression is proliferation dependent. A serum form of TK1 has been used as a biomarker in human medicine for many years and more recently to monitor canine lymphoma. Canine TK1 has not been cloned and studied. Therefore, dog and human TK1 cDNA were cloned and expressed, and the recombinant enzymes characterized. The serum and cellular forms of canine and human TK1 were studied by size-exclusion chromatography and the level of TK1 protein was determined using polyclonal and monoclonal anti-TK1 antibodies. Results Canine TK1 phosphorylated the thymidine (dThd analog 3'-azido-thymidine (AZT as efficiently as it did dThd, whereas AZT phosphorylation by human TK1 was less efficient than that of dThd. Dog TK1 was also more thermostable and pH tolerant than the human enzyme. Oligomeric forms were observed with both enzymes in addition to the tetrameric and dimeric forms. Cellular TK1 was predominantly seen in dimeric and tetrameric forms, in the case of both dog TK1 from MDCK cells and human TK1 from CEM cells. Active serum TK1 was found mainly in a high molecular weight form, and treatment with a reducing agent shifted the high molecular weight complex to lower molecular weight forms with reduced total activity. Western blot analysis demonstrated a polypeptide of 26 kDa (dog and 25 kDa (human for cellular and serum TK1. There was no direct correlation between serum TK1 activity and protein level. It appears that a substantial fraction of serum TK1 is not enzymatically active. Conclusions These results suggest that the serum TK1 protein differs from cellular or recombinant forms, is more active in high molecular weight complexes, and is sensitive to reducing agents. The results presented here provide important information for the future development and use of serum TK1 as a diagnostic biomarker in human and veterinary medicine.

  5. [Phosphorylation of glycogen synthase kinase-3beta induces epithelial mesenchymal transition in human peritoneal mesothelial cells].

    Science.gov (United States)

    Fan, Min; Liu, Fuyou; Yang, Yu; Ye, Yun; Huang, Guxiang

    2010-04-01

    To investigate the role of phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) inducing epithelial mesenchymal transition in human peritoneal mesothelial cells (HPMC). Primary HPMC was harvested from human omental tissue and maintained under defined in vitro conditions. The expression of p-GSK-3beta and total GSK-3beta in HMPC was detected by Western blot after incubation with different concentrations (0, 5, 10, 20, and 40 mmol/L)of LiCl at different time points (0, 1, 3, 6, and 12 h). The protein expression of E-cadherin and alpha-SMA was also examined after treatment with 20 mmol/L LiCl according to different time courses. The intracellular distribution and expression of alpha-SMA were determined by indirect immunofluorescence. LiCl stimulated phosphorylation of GSK-3beta and the effect was time-dependent and concentration-dependent to limited extent (PHMPC to epithelial mesenchymal transition and provides new clue for the treatment of peritoneal fibrosis.

  6. Choline kinase overexpression increases invasiveness and drug resistance of human breast cancer cells.

    Science.gov (United States)

    Shah, Tariq; Wildes, Flonne; Penet, Marie-France; Winnard, Paul T; Glunde, Kristine; Artemov, Dmitri; Ackerstaff, Ellen; Gimi, Barjor; Kakkad, Samata; Raman, Venu; Bhujwalla, Zaver M

    2010-07-01

    A direct correlation exists between increased choline kinase (Chk) expression, and the resulting increase of phosphocholine levels, and histological tumor grade. To better understand the function of Chk and choline phospholipid metabolism in breast cancer we have stably overexpressed one of the two isoforms of Chk-alpha known to be upregulated in malignant cells, in non-invasive MCF-7 human breast cancer cells. Dynamic tracking of cell invasion and cell metabolism were studied with a magnetic resonance (MR) compatible cell perfusion assay. The MR based invasion assay demonstrated that MCF-7 cells overexpressing Chk-alpha (MCF-7-Chk) exhibited an increase of invasion relative to control MCF-7 cells (0.84 vs 0.3). Proton MR spectroscopy studies showed significantly higher phosphocholine and elevated triglyceride signals in Chk overexpressing clones compared to control cells. A test of drug resistance in MCF-7-Chk cells revealed that these cells had an increased resistance to 5-fluorouracil and higher expression of thymidylate synthase compared to control MCF-7 cells. To further characterize increased drug resistance in these cells, we performed rhodamine-123 efflux studies to evaluate drug efflux pumps. MCF-7-Chk cells effluxed twice as much rhodamine-123 compared to MCF-7 cells. Chk-alpha overexpression resulted in MCF-7 human breast cancer cells acquiring an increasingly aggressive phenotype, supporting the role of Chk-alpha in mediating invasion and drug resistance, and the use of phosphocholine as a biomarker of aggressive breast cancers.

  7. Crystal Structure of Human Cyclin K, A Positive Regulator of Cyclin-Dependent Kinase 9

    Energy Technology Data Exchange (ETDEWEB)

    Baek,K.; Brown, R.; Birrane, G.; Ladias, J.

    2007-01-01

    K and the closely related cyclins T1, T2a, and T2b interact with cyclin-dependent kinase 9 (CDK9) forming multiple nuclear complexes, referred to collectively as positive transcription elongation factor b (P-TEFb). Through phosphorylation of the C-terminal domain of the RNA polymerase II largest subunit, distinct P-TEFb species regulate the transcriptional elongation of specific genes that play central roles in human physiology and disease development, including cardiac hypertrophy and human immunodeficiency virus-1 pathogenesis. We have determined the crystal structure of human cyclin K (residues 11-267) at 1.5 {angstrom} resolution, which represents the first atomic structure of a P-TEFb subunit. The cyclin K fold comprises two typical cyclin boxes with two short helices preceding the N-terminal box. A prominent feature of cyclin K is an additional helix (H4a) in the first cyclin box that obstructs the binding pocket for the cell-cycle inhibitor p27{sup Kip1}. Modeling of CDK9 bound to cyclin K provides insights into the structural determinants underlying the formation and regulation of this complex. A homology model of human cyclin T1 generated using the cyclin K structure as a template reveals that the two proteins have similar structures, as expected from their high level of sequence identity. Nevertheless, their CDK9-interacting surfaces display significant structural differences, which could potentially be exploited for the design of cyclin-targeted inhibitors of the CDK9-cyclin K and CDK9-cyclin T1 complexes.

  8. Rho kinase (ROK)-related proteins in human cavernous arteries: an immunohistochemical and functional approach.

    Science.gov (United States)

    Waldkirch, Eginhard S; Ückert, Stefan; Sohn, Michael; Kuczyk, Markus A; Hedlund, Petter

    2012-05-01

    Rho kinases (ROKs) cause calcium-independent modulation of smooth muscle contraction. A significant role for the RhoA/ROK pathway in mediating the contraction of the penile erectile tissue has been suggested. Moreover, it has been postulated that ROK activity might represent a key factor in the pathophysiology of erectile dysfunction. Up until today, little is known on the significance of ROK and related proteins in the control of blood flow in the corpus cavernosum. To investigate by means of immunohistochemistry and organ bath studies the significance of the Rho pathway in human cavernous arteries. The expression of ROK1, ROK2, RhoA, and RhoGDI in human cavernous arteries was investigated by means of immunohistochemistry; myographic studies were conducted in order to characterize the effects of the ROK inhibitor Y27632 on isolated cavernous arteries. Specimens of human cavernous arteries were processed for immunohistochemistry for ROK1, ROK2, RhoA, and RhoGDI. Circular penile vascular segments were mounted in a tissue bath and the effects of increasing concentrations of the ROK inhibitor Y27632 on the tension induced by norepinephrine (NE, 1 µM) were investigated. Alpha-actin immunoreactive cavernous arterioles also presented abundant staining specific for ROK1, ROK2, RhoA, and RhoGDI in the smooth musculature of the vascular wall. Cumulative addition of Y27632 dose-dependently reversed the tension induced by NE of isolated arterial segments. Y27632 produced relaxant responses with a reversion of tension of 34.3 ± 11.8% at a concentration of 1 µM. The findings are in support for a role of the Rho/ROK-mediated signaling in the regulation of muscle tone of human cavernous arteries. © 2012 International Society for Sexual Medicine.

  9. Targeting FMS-related tyrosine kinase receptor 3 with the human immunoglobulin G1 monoclonal antibody IMC-EB10.

    Science.gov (United States)

    Youssoufian, Hagop; Rowinsky, Eric K; Tonra, James; Li, Yiwen

    2010-02-15

    FMS-related tyrosine kinase receptor 3 (FLT3) is a class III receptor tyrosine kinase that holds considerable promise as a therapeutic target in hematologic malignancies. Current efforts directed toward the development of small-molecule tyrosine kinase inhibitors of FLT3 may be limited by off-target toxicities and the development of drug resistance. Target-specific antibodies could overcome these hurdles and provide additional mechanisms to enhance the antitumor efficacy of FLT3 inhibitors. IMC-EB10 is a novel antibody directed against FLT3. The binding of IMC-EB10 to FLT3 results in antiproliferative effects in vitro and in mouse models engrafted with human leukemia cells that harbor wild-type or constitutively activated FLT3. Future clinical trials will test these notions formally and will identify the most appropriate opportunities for this member of a new generation of antileukemic therapies. (c) 2010 American Cancer Society.

  10. KinMutRF: a random forest classifier of sequence variants in the human protein kinase superfamily.

    Science.gov (United States)

    Pons, Tirso; Vazquez, Miguel; Matey-Hernandez, María Luisa; Brunak, Søren; Valencia, Alfonso; Izarzugaza, Jose Mg

    2016-06-23

    The association between aberrant signal processing by protein kinases and human diseases such as cancer was established long time ago. However, understanding the link between sequence variants in the protein kinase superfamily and the mechanistic complex traits at the molecular level remains challenging: cells tolerate most genomic alterations and only a minor fraction disrupt molecular function sufficiently and drive disease. KinMutRF is a novel random-forest method to automatically identify pathogenic variants in human kinases. Twenty six decision trees implemented as a random forest ponder a battery of features that characterize the variants: a) at the gene level, including membership to a Kinbase group and Gene Ontology terms; b) at the PFAM domain level; and c) at the residue level, the types of amino acids involved, changes in biochemical properties, functional annotations from UniProt, Phospho.ELM and FireDB. KinMutRF identifies disease-associated variants satisfactorily (Acc: 0.88, Prec:0.82, Rec:0.75, F-score:0.78, MCC:0.68) when trained and cross-validated with the 3689 human kinase variants from UniProt that have been annotated as neutral or pathogenic. All unclassified variants were excluded from the training set. Furthermore, KinMutRF is discussed with respect to two independent kinase-specific sets of mutations no included in the training and testing, Kin-Driver (643 variants) and Pon-BTK (1495 variants). Moreover, we provide predictions for the 848 protein kinase variants in UniProt that remained unclassified. A public implementation of KinMutRF, including documentation and examples, is available online ( http://kinmut2.bioinfo.cnio.es ). The source code for local installation is released under a GPL version 3 license, and can be downloaded from https://github.com/Rbbt-Workflows/KinMut2 . KinMutRF is capable of classifying kinase variation with good performance. Predictions by KinMutRF compare favorably in a benchmark with other state

  11. Human Equilibrative Nucleoside Transporter 1, as a Predictor of 5-Fluorouracil Resistance in Human Pancreatic Cancer

    National Research Council Canada - National Science Library

    MASANORI TSUJIE; SHOJI NAKAMORI; SHIN NAKAHIRA; YUJI TAKAHASHI; NOBUYASU HAYASHI; JIRO OKAMI; HIROAKI NAGANO; KEIZO DONO; KOJI UMESHITA; MASATO SAKON; MORITO MONDEN

    2007-01-01

    ...€²-difluoro-deoxycytidine) sensitivity in pancreatic cancer. Materials and Methods: The relationship between 5-FU and gemcitabine sensitivity and the mRNA levels of human equilibrative nucleoside transporter 1 (hENT1...

  12. The human biliverdin reductase-based peptide fragments and biliverdin regulate protein kinase Cδ activity: the peptides are inhibitors or substrate for the protein kinase C.

    Science.gov (United States)

    Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E M; Tudor, Cicerone; Hagen, Fred K; Maines, Mahin D

    2012-07-13

    PKCδ, a Ser/Thr kinase, promotes cell growth, tumorigenesis, and apoptosis. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase, inhibits apoptosis by reducing biliverdin-IX to antioxidant bilirubin. The enzymes are activated by similar stimuli. Reportedly, hBVR is a kinase-independent activator of PKCδ and is transactivated by the PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C., and Maines, M. D. (2012) J. Biol. Chem. 287, 1066-1079). Presently, we examined interactions between the two proteins in the context of regulation of their activities and defining targets of hBVR phosphorylation by PKCδ. LC-MS/MS analysis of PKCδ-activated intact hBVR identified phosphorylated serine positions 21, 33, 230, and 237, corresponding to the hBVR Src homology-2 domain motif (Ser(230) and Ser(237)), flanking the ATP-binding motif (Ser(21)) and in PHPS sequence (Ser(33)) as targets of PKCδ. Ser(21) and Ser(230) were also phosphorylated in hBVR-based peptides. The Ser(230)-containing peptide was a high affinity substrate for PKCδ in vitro and in cells; the relative affinity was PKCδ > PKCβII > PKCζ. Two overlapping peptides spanning this substrate, KRNRYLSF and SFHFKSGSL, were effective inhibitors of PKCδ kinase activity and PKCδ-supported activation of transcription factors Elk1 and NF-κB. Only SFHFKSGSL, in PKCδ-transfected phorbol 12-myristate 13-acetate-stimulated cells, caused membrane blebbing and cell loss. Biliverdin noncovalently inhibited PKCδ, whereas PKCδ potentiated hBVR reductase activity and accelerated the rate of bilirubin formation. This study, together with previous findings, reveals an unexpected regulatory interplay between PKCδ and hBVR in modulating cell death/survival in response to various activating stimuli. In addition, this study has identified novel substrates for and inhibitors of PKCδ. We suggest that hBVR-based technology may have utility to modulate PKCδ-mediated functions in the cell.

  13. Biodentine induces human dental pulp stem cell differentiation through mitogen-activated protein kinase and calcium-/calmodulin-dependent protein kinase II pathways.

    Science.gov (United States)

    Luo, Zhirong; Kohli, Meetu R; Yu, Qing; Kim, Syngcuk; Qu, Tiejun; He, Wen-xi

    2014-07-01

    Biodentine (Septodont, Saint-Maur-des-Fossès, France), a new tricalcium silicate cement formulation, has been introduced as a bioactive dentine substitute to be used in direct contact with pulp tissue. The aim of this study was to investigate the response of human dental pulp stem cells (hDPSCs) to the material and whether mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and calcium-/calmodulin-dependent protein kinase II (CaMKII) signal pathways played a regulatory role in Biodentine-induced odontoblast differentiation. hDPCs obtained from impacted third molars were incubated with Biodentine. Odontoblastic differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time reverse-transcriptase polymerase chain reaction for the analysis of messenger RNA expression of the following differentiation gene markers: osteocalcin (OCN), dentin sialophosprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). Cell cultures in the presence of Biodentine were exposed to specific inhibitors of MAPK (U0126, SB203580, and SP600125), NF-κB (pyrrolidine dithiocarbamate), and CaMKII (KN-93) pathways to evaluate the regulatory effect on the expression of these markers and mineralization assay. Biodentine significantly increased alkaline phosphatase activity and mineralized nodule formation and the expression of OCN, DSPP, DMP1, and BSP. The MAPK inhibitor for extracellular signal-regulated kinase 1/2 (U0126) and Jun N-terminal kinase (SP600125) significantly decreased the Biodentine-induced mineralized differentiation of hDPSCs and OCN, DSPP, DMP1, and BSP messenger RNA expression, whereas p38 MAPK inhibitors (SB203580) had no effect. The CaMKII inhibitor KN-93 significantly attenuated and the NF-κB inhibitor pyrrolidine dithiocarbamate further enhanced the up-regulation of Biodentine-induced gene expression and mineralization. Biodentine is a bioactive and biocompatible material capable

  14. Inhibition of mitogen-activated protein kinase kinase enhances apoptosis induced by arsenic trioxide in human breast cancer MCF-7 cells.

    Science.gov (United States)

    Ye, Jian; Li, Aiping; Liu, Qizhan; Wang, Xinru; Zhou, Jianwei

    2005-12-01

    Arsenic trioxide (As2O3) has recently been used to treat acute promyelocytic leukaemia and has activity in vitro against several solid tumour cell lines where the induction of differentiation and apoptosis are the prime effects. The mechanism of As2O3-induced cell death has yet to be clarified, especially in solid cancers. In the present study, the human breast cancer cell line MCF-7 was examined as a cellular model for As2O3 treatment. The involvement of extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) was investigated in As2O3-induced cell death. 3. It was found that As2O3 activates the prosurvival mitogen-activated protein kinase kinase (MEK)/ERK pathway in MCF-7 cells, which, conversely, may compromise the efficacy of As2O3. Hence, a combination treatment of As2O3 and MEK inhibitors was investigated to determine whether this treatment could lead to enhanced growth inhibition and apoptosis in MCF-7 cells. 4. Inhibition of MEK/ERK with the pharmacological inhibitors U0126 (10 micromol/L) or PD98059 (20 micromol/L) together with As2O3 (2 and 5 micromol/L) resulted in a significant enhancement of growth inhibition in breast cancer MCF-7 cells as determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and [Methyl-3H]-thymidine incorporation. Furthermore, the results demonstrated that combined treatment with As2O3 and the MEK1/2 inhibitor U0126 could augment breast cancer MCF-7 cell apoptosis approximately twofold compared with the effects of the two drugs alone, as determined by Hoechst 33258 or annexin V/propidium iodide (PI) staining and flow cytometry. 5. In addition, As2O3 activated p38 in a dose-dependent manner, but had no effect on JNK1/2. Treatment with a p38 inhibitor did not prevent As2O3-induced apoptosis. 6. In conclusion, the results of the present study showed that enhanced apoptosis is detected in breast cancer MCF-7 cells in the presence of As2O3 and an MEK inhibitor, which may be a

  15. Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38.

    Science.gov (United States)

    Yustein, Jason T; Xia, Liang; Kahlenburg, J Michelle; Robinson, Dan; Templeton, Dennis; Kung, Hsing-Jien

    2003-09-18

    The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated. Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s. Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken). For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively. These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus. All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase. We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association. Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence.

  16. Monoclonal Antibodies to the Human Insulin Receptor that Activate Glucose Transport but not Insulin Receptor Kinase Activity

    Science.gov (United States)

    Forsayeth, John R.; Caro, Jose F.; Sinha, Madhur K.; Maddux, Betty A.; Goldfine, Ira D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  17. MNADK, a Long-Awaited Human Mitochondrion-Localized NAD Kinase.

    Science.gov (United States)

    Zhang, Ren

    2015-08-01

    Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, play essential roles in numerous cellular processes in all organisms. NADP maintains a pool of its reducing equivalent, NADPH, which regenerates cellular oxidative defense systems to counteract oxidative damages. Mitochondria represent a major source of oxidative stress, because the majority of superoxide, a reactive oxygen species, is generated from the mitochondrial respiratory chain. Therefore, as universal electron carriers in cellular electron transfer reactions, the pyridine nucleotides are required by mitochondria for both antioxidant protection and biosynthetic pathways. The NAD kinase (NADK) is the sole NADP biosynthetic enzyme. Because NADP is membrane-impermeable, eukaryotes need compartment-specific NADKs for different organelles. Consistently, in both yeast and plants, three compartment-specific NADKs have been identified. In contrast, even though the first human NADK, a cytosolic one, was identified in 2001, the identity of a hypothesized mitochondrial NADK remained elusive, until a recent discovery that the uncharacterized human gene C5ORF33 encodes a mitochondrion-localized NADK, referred to as MNADK. Three groups have characterized MNADK functions based on distinct systems involving yeast, mouse, and human studies, from aspects of both in vitro and in vivo evidence. MNADK is a mitochondrial NADK that is enriched and nutritionally-regulated in mouse liver, and a MNADK-deficient patient exhibits symptoms characteristic of mitochondrial disease. The identification of MNADK provides a key clue to the mechanism involved in mitochondrial NADPH production and the maintenance of redox balance in mammalian cells. The roles of MNADK in physiological and pathological processes have yet to be discovered. © 2015 Wiley Periodicals, Inc.

  18. Aspirin augments IgE-mediated histamine release from human peripheral basophils via Syk kinase activation.

    Science.gov (United States)

    Matsuo, Hiroaki; Yokooji, Tomoharu; Morita, Hironobu; Ooi, Mina; Urata, Kana; Ishii, Kaori; Takahagi, Shunsuke; Yanase, Yuhki; Hiragun, Takaaki; Mihara, Shoji; Hide, Michihiro

    2013-12-01

    Non-steroidal anti-inflammatory drugs (NSAIDs), especially aspirin, and food additives (FAs) may exacerbate allergic symptoms in patients with chronic idiopathic urticaria and food-dependent exercise-induced anaphylaxis (FDEIA). Augmentation of histamine release from human mast cells and basophils by those substances is speculated to be the cause of exacerbated allergic symptoms. We sought to investigate the mechanism of action of aspirin on IgE-mediated histamine release. The effects of NSAIDs, FAs or cyclooxygenase (COX) inhibitors on histamine release from human basophils concentrated by gravity separation were evaluated. Benzoate and tartrazine, which have no COX inhibitory activity, augmented histamine release from basophils similar to aspirin. In contrast, ibuprofen, meloxicam, FR122047 and NS-398, which have COX inhibitory activity, did not affect histamine release. These results indicate that the augmentation of histamine release by aspirin is not due to COX inhibition. It was observed that aspirin augmented histamine release from human basophils only when specifically activated by anti-IgE antibodies, but not by A23187 or formyl-methionyl-leucyl-phenylalanine. When the IgE receptor signaling pathway was activated, aspirin increased the phosphorylation of Syk. Moreover, patients with chronic urticaria and FDEIA tended to be more sensitive to aspirin as regards the augmentation of histamine release, compared with healthy controls. Aspirin enhanced histamine release from basophils via increased Syk kinase activation, and that the augmentation of histamine release by NSAIDs or FAs may be one possible cause of worsening symptoms in patients with chronic urticaria and FDEIA.

  19. Reishi immuno-modulation protein induces interleukin-2 expression via protein kinase-dependent signaling pathways within human T cells.

    Science.gov (United States)

    Hsu, Hsien-Yeh; Hua, Kuo-Feng; Wu, Wei-Chi; Hsu, Jason; Weng, Shih-Ting; Lin, Tsai-Leng; Liu, Chun-Yi; Hseu, Ruey-Shyang; Huang, Ching-Tsan

    2008-04-01

    Ganoderma lucidum, a medicinal fungus is thought to possess and enhance a variety of human immune functions. An immuno-modulatory protein, Ling Zhi-8 (LZ-8) isolated from G. lucidum exhibited potent mitogenic effects upon human peripheral blood lymphocytes (PBL). However, LZ-8-mediated signal transduction in the regulation of interleukin-2 (IL-2) gene expression within human T cells is largely unknown. Here we cloned the LZ-8 gene of G. lucidum, and expressed the recombinant LZ-8 protein (rLZ-8) by means of a yeast Pichia pastoris protein expression system. We found that rLZ-8 induces IL-2 gene expression via the Src-family protein tyrosine kinase (PTK), via reactive oxygen species (ROS), and differential protein kinase-dependent pathways within human primary T cells and cultured Jurkat T cells. In essence, we have established the nature of the rLZ-8-mediated signal-transduction pathways, such as PTK/protein kinase C (PKC)/ROS, PTK/PLC/PKCalpha/ERK1/2, and PTK/PLC/PKCalpha/p38 pathways in the regulation of IL-2 gene expression within human T cells. Our current results of analyzing rLZ-8-mediated signal transduction in T cells might provide a potential application for rLZ-8 as a pharmacological immune-modulating agent. (c) 2008 Wiley-Liss, Inc.

  20. Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

    Science.gov (United States)

    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

    2012-08-01

    Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

  1. Structure of protein kinase CK2: dimerization of the human beta-subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Mietens, U; Issinger, O G

    1996-01-01

    Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a heterotetrameric serine-threonine kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits. Us...

  2. Nucleotide binding to human UMP-CMP kinase using fluorescent derivatives -- a screening based on affinity for the UMP-CMP binding site.

    Science.gov (United States)

    Topalis, Dimitri; Kumamoto, Hiroki; Amaya Velasco, Maria-Fernanda; Dugué, Laurence; Haouz, Ahmed; Alexandre, Julie Anne C; Gallois-Montbrun, Sarah; Alzari, Pedro Maria; Pochet, Sylvie; Agrofoglio, Luigi André; Deville-Bonne, Dominique

    2007-07-01

    Methylanthraniloyl derivatives of ATP and CDP were used in vitro as fluorescent probes for the donor-binding and acceptor-binding sites of human UMP-CMP kinase, a nucleoside salvage pathway kinase. Like all NMP kinases, UMP-CMP kinase binds the phosphodonor, usually ATP, and the NMP at different binding sites. The reaction results from an in-line phosphotransfer from the donor to the acceptor. The probe for the donor site was displaced by the bisubstrate analogs of the Ap5X series (where X = U, dT, A, G), indicating the broad specificity of the acceptor site. Both CMP and dCMP were competitors for the acceptor site probe. To find antimetabolites for antivirus and anticancer therapies, we have developed a method of screening acyclic phosphonate analogs that is based on the affinity of the acceptor-binding site of the human UMP-CMP kinase. Several uracil vinylphosphonate derivatives had affinities for human UMP-CMP kinase similar to those of dUMP and dCMP and better than that of cidofovir, an acyclic nucleoside phosphonate with a broad spectrum of antiviral activities. The uracil derivatives were inhibitors rather than substrates of human UMP-CMP kinase. Also, the 5-halogen-substituted analogs inhibited the human TMP kinase less efficiently. The broad specificity of the enzyme acceptor-binding site is in agreement with a large substrate-binding pocket, as shown by the 2.1 A crystal structure.

  3. Effect of oncogene activating mutations and kinase inhibitors on amino acid metabolism of human isogenic breast cancer cells.

    Science.gov (United States)

    Kim, Eung-Sam; Samanta, Animesh; Cheng, Hui Shan; Ding, Zhaobing; Han, Weiping; Toschi, Luisella; Chang, Young Tae

    2015-12-01

    We investigated the changes in amino acid (AA) metabolism induced in MCF10A, a human mammary epithelial cell line, by the sequential knock-in of K-Ras and PI3K mutant oncogenes. Differentially regulated genes associated to AA pathways were identified on comparing gene expression patterns in the isogenic cell lines. Additionally, we monitored the changes in the levels of AAs and transcripts in the cell lines treated with kinase inhibitors (REGO: a multi-kinase inhibitor, PI3K-i: a PI3K inhibitor, and MEK-i: a MEK inhibitor). In total, 19 AAs and 58 AA-associated transcripts were found to be differentially regulated by oncogene knock-in and by drug treatment. In particular, the multi-kinase and MEK inhibitor affected pathways in K-Ras mutant cells, whereas the PI3K inhibitor showed a major impact in the K-Ras/PI3K double mutant cells. These findings may indicate the dependency of AA metabolism on the oncogene mutation pattern in human cancer. Thus, future therapy might include combinations of kinase inhibitors and drug targeting enzymes of AA pathways.

  4. Equitoxic doses of 5-azacytidine and 5-aza-2'deoxycytidine induce diverse immediate and overlapping heritable changes in the transcriptome

    DEFF Research Database (Denmark)

    Qiu, Xiangning; Hother, Christoffer; Ralfkiær, Ulrik M

    2010-01-01

    The hypomethylating agent 5-Azacytidine (5-Aza-CR) is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS). Surprisingly, the deoxyribonucleoside analog 5-Aza-2'deoxycytidine (5-Aza-CdR) did not have a similar effect on survival in a large clinical trial. Both...

  5. Dietary exposure to di-isobutyl phthalate increases urinary 5-methyl-2'-deoxycytidine level and affects reproductive function in adult male mice.

    Science.gov (United States)

    Pan, Yitao; Wang, Xiaoyang; Yeung, Leo W Y; Sheng, Nan; Cui, Qianqian; Cui, Ruina; Zhang, Hongxia; Dai, Jiayin

    2017-11-01

    Phthalates are a large family of ubiquitous environmental pollutants suspected of being endocrine disruptors. Epidemiological studies have associated phthalate metabolites with decreased reproductive parameters and linked phthalate exposure with the level of urinary 5-methyl-2'-deoxycytidine (5mdC, a product of methylated DNA). In this study, adult male mice were exposed to 450mg di-isobutyl phthalate (DiBP)/(kg·day) via dietary exposure for 28days. Mono-isobutyl phthalate (MiBP, the urinary metabolite) and reproductive function parameters were determined. The levels of 5mdC and 5-hydroxymethyl-2'-deoxycytidine (5hmdC) were measured in urine to evaluate if their contents were also altered by DiBP exposure in this animal model. Results showed that DiBP exposure led to a significant increase in the urinary 5mdC level and significant decreases in sperm concentration and motility in the epididymis, accompanied with reduced testosterone levels and down-regulation of the P450 cholesterol side-chain cleavage enzyme (P450scc) gene in the mice testes. Our findings indicated that exposure to DiBP increased the urinary 5mdC levels, which supported our recent epidemiological study about the associations of urinary 5mdC with phthalate exposure in the male human population. In addition, DiBP exposure impaired male reproductive function, possibly by disturbing testosterone levels; P450scc might be a major steroidogenic enzyme targeted by DiBP or other phthalates. Copyright © 2017. Published by Elsevier B.V.

  6. Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family.

    Directory of Open Access Journals (Sweden)

    Douglas R Boettner

    2008-01-01

    Full Text Available Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK, was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i incubation of ameba with anti-PATMK antibodies; (ii PATMK mRNA knock-down using a novel shRNA expression system; and (iii expression of a carboxy-truncation of PATMK (PATMK(delta932. Expression of the carboxy-truncation of PATMK(delta932 also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

  7. Identification of Human UMP/CMP Kinase 1 as Doxorubicin Binding Target Using Protein Microarray.

    Science.gov (United States)

    Chen, Shuxian; Wang, Xu; Ye, Xianghui; Ma, Donghui; Chen, Caiwei; Cai, Junlong; Fu, Yongfeng; Cheng, Xunjia; Chen, Yun; Gong, Xiaohai; Jin, Jian

    2017-09-01

    Doxorubicin (DOX) is a leading anthracycline drug with exceptional efficacy; however, little is known about the molecular mechanisms of its side effects, which include heart muscle damage, noncancerous cell death, and drug resistance. A total of 17,950 human proteins expressed in HEK293 cells were screened and yielded 14 hits. Competitive and binding experiments further verified the binding of DOX to UMP/CMP kinase 1 (CMPK1), and microscale thermophoresis showed that DOX binds to CMPK1 with a Kd of 1216 nM. In addition, we observed that the binding of DOX to CMPK1 activated the phosphorylation of CMP, dCMP, and UMP. A significant activation was observed at the concentration of 30 µM DOX and reached plateau at the concentration of DOX 30 µM, 150 µM, and 100 µM, respectively. DOX would add up stimulation of CMPK1 by DTT and overcome inhibition of CMPK1 by NaF, EDTA. In summary, we showed that DOX might bind to the nonactive site of CMPK1 and regulate its activity with magnesium.

  8. Human TFIIH Kinase CDK7 Regulates Transcription-Associated Chromatin Modifications

    Directory of Open Access Journals (Sweden)

    Christopher C. Ebmeier

    2017-08-01

    Full Text Available CDK7 phosphorylates the RNA polymerase II (pol II C-terminal domain CTD and activates the P-TEFb-associated kinase CDK9, but its regulatory roles remain obscure. Here, using human CDK7 analog-sensitive (CDK7as cells, we observed reduced capping enzyme recruitment, increased pol II promoter-proximal pausing, and defective termination at gene 3′ ends upon CDK7 inhibition. We also noted that CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5′ ends and H3K36me3 was displaced toward gene 3′ ends in CDK7as cells. Mass spectrometry identified factors that bound TFIIH-phosphorylated versus P-TEFb-phosphorylated CTD (versus unmodified; capping enzymes and H3K4 methyltransferase complexes, SETD1A/B, selectively bound phosphorylated CTD, and the H3K36 methyltransferase SETD2 specifically bound P-TEFb-phosphorylated CTD. Moreover, TFIIH-phosphorylated CTD stimulated SETD1A/B activity toward nucleosomes, revealing a mechanistic basis for CDK7 regulation of H3K4me3 spreading. Collectively, these results implicate a CDK7-dependent “CTD code” that regulates chromatin marks in addition to RNA processing and pol II pausing.

  9. Characterization of protein kinase C and its isoforms in human T lymphocytes.

    Science.gov (United States)

    Beyers, A D; Hanekom, C; Rheeder, A; Strachan, A F; Wooten, M W; Nel, A E

    1988-11-15

    Protein kinase C (PKC) regulates numerous T cell functions and is present in abundance in normal human T cells and certain T cell lines. Although crude Triton X-100 soluble material obtained from T cell pellets contains minimal PKC activity, DEAE chromatography revealed that 12 to 37% of cellular PKC was membrane associated, probably due to removal of an inhibitor through column chromatography. As in other tissues, PKC from lymphoid tissue was phospholipid and Ca2+ dependent and diolein reduced the Ca2+ requirements for enzyme activity. Hydroxylapatite chromatography revealed that T cells possess two major peaks of PKC activity. Although, the enzyme in these peaks had similar m.w. and identical iso-electric mobility, the proteins differed with respect to their autophosphorylation sites and immunoreactivity toward an isoform specific antibody. Furthermore, differences in their activities in the presence of phospholipid, diolein, and limiting amounts of Ca2+ imply that these isoforms may be differentially activated. We discuss optimal conditions for activation of PKC and its isoforms for study of T lymphocyte cellular function.

  10. P21-activated kinase 1 regulates resistance to BRAF inhibition in human cancer cells.

    Science.gov (United States)

    Babagana, Mahamat; Johnson, Sydney; Slabodkin, Hannah; Bshara, Wiam; Morrison, Carl; Kandel, Eugene S

    2017-05-01

    BRAF is a commonly mutated oncogene in various human malignancies and a target of a new class of anti-cancer agents, BRAF-inhibitors (BRAFi). The initial enthusiasm for these agents, based on the early successes in the management of metastatic melanoma, is now challenged by the mounting evidence of intrinsic BRAFi-insensitivity in many BRAF-mutated tumors, by the scarcity of complete responses, and by the inevitable emergence of drug resistance in initially responsive cases. These setbacks put an emphasis on discovering the means to increase the efficacy of BRAFi and to prevent or overcome BRAFi-resistance. We explored the role of p21-activated kinases (PAKs), in particular PAK1, in BRAFi response. BRAFi lowered the levels of active PAK1 in treated cells. An activated form of PAK1 conferred BRAFi-resistance on otherwise sensitive cells, while genetic or pharmacologic suppression of PAK1 had a sensitizing effect. While activation of AKT1 and RAC1 proto-oncogenes increased BRAFi-tolerance, the protective effect was negated in the presence of PAK inhibitors. Furthermore, combining otherwise ineffective doses of PAK- and BRAF-inhibitors synergistically affected intrinsically BRAFi-resistant cells. Considering the high incidence of PAK1 activation in cancers, our findings suggests PAK inhibition as a strategy to augment BRAFi therapy and overcome some of the well-known resistance mechanisms. © 2017 Wiley Periodicals, Inc.

  11. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, C.W.; Connelly, M.A.; Zhang, H.; Sipley, J.A. [Brookhaven National Lab., Upton, NY (United States). Biology Dept.; Lees-Miller, S.P.; Lintott, L.G. [Univ. of Calgary, Alberta (Canada). Dept. of Biological Sciences; Sakaguchi, Kazuyasu; Appella, E. [National Institutes of Health, Bethesda, MD (United States). Lab. of Cell Biology

    1994-11-05

    Although much has been learned about the structure and function of p53 and the probable sequence of subsequent events that lead to cell cycle arrest, little is known about how DNA damage is detected and the nature of the signal that is generated by DNA damage. Circumstantial evidence suggests that protein kinases may be involved. In vitro, human DNA-PK phosphorylates a variety of nuclear DNA-binding, regulatory proteins including the tumor suppressor protein p53, the single-stranded DNA binding protein RPA, the heat shock protein hsp90, the large tumor antigen (TAg) of simian virus 40, a variety of transcription factors including Fos, Jun, serum response factor (SRF), Myc, Sp1, Oct-1, TFIID, E2F, the estrogen receptor, and the large subunit of RNA polymerase II (reviewed in Anderson, 1993; Jackson et al., 1993). However, for most of these proteins, the sites that are phosphorylated by DNA-PK are not known. To determine if the sites that were phosphorylated in vitro also were phosphorylated in vivo and if DNA-PK recognized a preferred protein sequence, the authors identified the sites phosphorylated by DNA-PK in several substrates by direct protein sequence analysis. Each phosphorylated serine or threonine is followed immediately by glutamine in the polypeptide chain; at no other positions are the amino acid residues obviously constrained.

  12. Ganciclovir uptake in human mammary carcinoma cells expressing herpes simplex virus thymidine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Haberkorn, Uwe; Khazaie, Khashayarsha; Morr, Iris; Altmann, Annette; Mueller, Markus; Kaick, Gerhard van

    1998-05-01

    Assessment of suicide enzyme activity would have considerable impact on the planning and the individualization of suicide gene therapy of malignant tumors. This may be done by determining the pharmacokinetics of specific substrates. We generated ganciclovir (GCV)-sensitive human mammary carcinoma cell lines after transfection with a retroviral vector bearing the herpes simplex virus thymidine kinase (HSV-tk) gene. Thereafter, uptake measurements and HPLC analyses were performed up to 48 h in an HSV-tk-expressing cell line and in a wild-type cell line using tritiated GCV. HSV-tk-expressing cells showed higher GCV uptake and phosphorylation than control cells, whereas in wild-type MCF7 cells no phosphorylated GCV was detected. In bystander experiments the total GCV uptake was related to the amount of HSV-tk-expressing cells. Furthermore, the uptake of GCV correlated closely with the growth inhibition (r=0.92). Therefore, the accumulation of specific substrates may serve as an indicator of the HSV-tk activity and of therapy outcome. Inhibition and competition experiments demonstrated slow transport of GCV by the nucleoside carriers. The slow uptake and low affinity to HSV-tk indicate that GCV is not an ideal substrate for the nucleoside transport systems or for HSV-tk. This may be the limiting factor for therapy success, necessitating the search for better substrates of HSV-tk.

  13. Human NIMA-related kinase 6 is one of the Fe65 WW domain binding proteins.

    Science.gov (United States)

    Lee, Eun Jeoung; Hyun, Sung Hee; Chun, Jaesun; Kang, Sang Sun

    2007-07-06

    The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we present evidence that human Nek6 is associated with Fe65. Based on the presence of Fe65 WW domain binding motifs ((267)PPLP(270)) in the Nek6 catalytic domain, we observed that Nek6 interacts physically with Fe65 both in vivo and in vitro, using a pull-down approach. Additionally, we detected co-localization of Nek6 and Fe65 via confocal microscopy. Co-localization of Nek6 and Fe65 was disrupted by mutation of the WW domain binding motifs ((267)PPLP(270)). Finally, when transient transfection assays were performed, interaction of Nek6 (wt) with Fe65 induced substantial cell apoptosis, whereas interaction using the Nek6 pplp mutant ((267)PPLP(270) changes (267)APVA(270)) did not. Thus, our observations indicated that Nek6 binds to Fe65 through its (267)PPLP(270) motif and that the protein-protein interaction between Nek6 and Fe65 regulates their subcellular localization and cell apoptosis.

  14. Lck is involved in interleukin-2 induced proliferation but not cell survival in human T cells through a MAP kinase-independent pathway

    DEFF Research Database (Denmark)

    Brockdorff, J; Nielsen, M; Kaltoft, K

    2000-01-01

    The role of Lck in IL-2-induced proliferation and cell survival is still controversial. Here, we show that the Src family kinase inhibitor, PP1, reduced the IL-2-induced proliferation of human T cells significantly without inhibiting the anti-apoptotic effect of IL-2. As Lck is the only Src family...... kinase activated upon IL-2 stimulation in T cells, this indicates that Lck is involved in IL-2-induced proliferation but not survival. IL-2-induced MAP kinase activation was only slightly inhibited by PP1, suggesting that Lck is not essential for IL-2-induced MAP kinase activation in human T cells. We...... found that an IL-2-sensitive, human mycosis fungoides-derived tumor T cell line is Lck negative, and that the IL-2-induced MAP kinase activation is comparable to non-cancerous T cells, although a little delayed in kinetics. An Lck expressing clone was established by transfecting Lck into mycosis...

  15. A Dual Role for the Nonreceptor Tyrosine Kinase Pyk2 during the Intracellular Trafficking of Human Papillomavirus 16.

    Science.gov (United States)

    Gottschalk, Elinor Y; Meneses, Patricio I

    2015-09-01

    The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study advances our knowledge of

  16. A novel role of protein tyrosine kinase2 in mediating chloride secretion in human airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Lihua Liang

    Full Text Available Ca(2+ activated Cl(- channels (CaCC are up-regulated in cystic fibrosis (CF airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl(- secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca(2+, which not only activates epithelial CaCCs, but also inhibits epithelial Na(+ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl(- secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2 is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca(2+ and Cl(- secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.

  17. MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein tyrosine and serine/threonine kinases

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Johansen, B

    1999-01-01

    B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h...... post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine....../threonine kinases in MHC-I-mediated apoptosis in human B-cells and suggest the presence of several MHC-I signaling pathways leading to diverse effects in these cells....

  18. The Role of Sphingosine Kinase 2 in Apoptosis of Human Breast Cancer Cells

    National Research Council Canada - National Science Library

    Sankala, Heidi; Spiegel, Sarah

    2006-01-01

    .... However, whether it also has an intracellular action is still a matter of debate. S1P is formed by the ATP-dependent phosphorylation of sphingosine catalyzed by types 1 and 2 sphingosine kinase (SphK...

  19. The Role of Sphingosine Kinase 2 in Apoptosis of Human Breast Cancer

    National Research Council Canada - National Science Library

    Sankala, Heidi; Spiegel, Sarah

    2005-01-01

    .... However, whether it also has an intracellular action is still a matter of debate. S1P is formed by the ATP-dependent phosphorylation of sphingosine catalyzed by types 1 and 2 sphingosine kinase (SphK...

  20. Aspirin Augments IgE-Mediated Histamine Release from Human Peripheral Basophils via Syk Kinase Activation

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsuo

    2013-01-01

    Conclusions: Aspirin enhanced histamine release from basophils via increased Syk kinase activation, and that the augmentation of histamine release by NSAIDs or FAs may be one possible cause of worsening symptoms in patients with chronic urticaria and FDEIA.

  1. The effects of knockdown of rho-associated kinase 1 and zipper-interacting protein kinase on gene expression and function in cultured human arterial smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Jing-Ti Deng

    Full Text Available Rho-associated kinase (ROCK and zipper-interacting protein kinase (ZIPK have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC. Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated. IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude

  2. Deoxyribonucleoside kinases activate nucleoside antibiotics in severely pathogenic bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Shannon, O.; Clausen, A.R.

    2007-01-01

    pathogenic staphylococci and streptococci. We show that pyrimidine-based nucleoside analogs, like 3'-azido-3'-deoxythymidine (AZT) and 2',2'-difluoro-2'deoxycytidine (gemcitabine), are specifically activated by the endogenous bacterial deoxyribonucleoside kinases, leading to cell death. Deoxyribonucleoside...... kinase-deficient Escherichia coli strains become highly susceptible to nucleoside analogs when they express recombinant kinases from Staphylococcus aureus or Streptococcus pyogenes. We further demonstrate that recombinant S. aureus deoxyadenosine kinase efficiently phosphorylates the anticancer drug...... gemcitabine in vitro and is therefore the key enzyme in the activation pathway. When adult mice were infected intraperitoneally with a fatal dose of S. pyogenes strain AP1 and afterwards received gemcitabine, they failed to develop a systemic infection. Nucleoside analogs may therefore represent a promising...

  3. Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

    Science.gov (United States)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2004-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

  4. HaloTag-based purification of functional human kinases from mammalian cells.

    Science.gov (United States)

    Ohana, Rachel Friedman; Hurst, Robin; Vidugiriene, Jolanta; Slater, Michael R; Wood, Keith V; Urh, Marjeta

    2011-04-01

    Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

  5. Glycogen synthase kinase-3beta regulates differentiation-induced apoptosis of human neural progenitor cells.

    Science.gov (United States)

    Jaeger, Alexandra; Baake, Jana; Weiss, Dieter G; Kriehuber, Ralf

    2013-02-01

    Glycogen synthase kinase-3beta is a multifunctional key regulator enzyme in neural developmental processes and a main component of the canonical Wnt signaling pathway. It is already known that the Wnt-driven differentiation of neural progenitor cells is accompanied by an increase of apoptosis at which the pro-apoptotic function of GSK-3beta is still discussed. The aim of the present study was to investigate whether the phosphorylation level of GSK-3beta at serine 9 is the primary regulatory mechanism of differentiation-induced apoptosis. Differentiating human neural ReNcell VM progenitor cells were treated with the specific GSK-3beta inhibitor SB216763 (10 μM) and analyzed in respect to the intrinsic apoptosis pathway regulation using microscopy and protein expression analysis. Differentiation of ReNcell VM cells was accompanied by cell morphological changes, cytoskeleton rearrangement and apoptosis increase. Treatment of differentiating cells with SB216763 induced a significant dephosphorylation of GSK-3beta at serine 9 accompanied by a significant decrease of apoptosis of about 0.7±0.03% and reduced activation of caspase-3 as well as BAX and PARP cleavage during the first 12h of differentiation compared to untreated, differentiating cells. Dephosphorylation of GSK-3beta at serine 9 appears not solely to be responsible for its pro-apoptotic function, because we observed a decrease of intrinsic apoptosis after treatment of the cells with the specific GSK-3beta inhibitor SB216763. We assume that GSK-3beta drives neural progenitor cell apoptosis by direct interaction with pro-apoptotic BAX or by indirect influence on the canonical Wnt/beta-catenin target gene transcription. Copyright © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.

  6. Activin-like kinase 2 functions in peri-implantation uterine signaling in mice and humans.

    Directory of Open Access Journals (Sweden)

    Caterina Clementi

    2013-11-01

    Full Text Available Implantation of a blastocyst in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP ligands and receptors are expressed in the uterus of pregnant mice, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2, during mouse pregnancy by producing mice carrying a conditional ablation of Alk2 in the uterus (Alk2 cKO mice. In the absence of ALK2, embryos demonstrate delayed invasion into the uterine epithelium and stroma, and upon implantation, stromal cells fail to undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein β (Cebpb expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of CEBPB in the stroma to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (hESC and discovered that ablation of ALK2 alters hESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP analysis of decidualizing hESC confirmed that BMP signaling proteins, SMAD1/5, directly regulate expression of CEBPB by binding a distinct regulatory sequence in the 3' UTR of this gene; CEBPB, in turn, regulates the expression of progesterone receptor (PGR. Our work clarifies the conserved mechanisms through which BMPs regulate peri-implantation in rodents and primates and, for the first time, uncovers a linear pathway of BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization.

  7. Thermodynamics parameters for binding of halogenated benzotriazole inhibitors of human protein kinase CK2α.

    Science.gov (United States)

    Winiewska, Maria; Kucińska, Katarzyna; Makowska, Małgorzata; Poznański, Jarosław; Shugar, David

    2015-10-01

    The interaction of human CK2α (hCK2α) with nine halogenated benzotriazoles, TBBt and its analogues representing all possible patterns of halogenation on the benzene ring of benzotriazole, was studied by biophysical methods. Thermal stability of protein-ligand complexes, monitored by calorimetric (DSC) and optical (DSF) methods, showed that the increase in the mid-point temperature for unfolding of protein-ligand complexes (i.e. potency of ligand binding to hCK2α) follow the inhibitory activities determined by biochemical assays. The dissociation constant for the ATP-hCK2α complex was estimated with the aid of microscale thermophoresis (MST) as 4.3±1.8 μM, and MST-derived dissociation constants determined for halogenated benzotriazoles, when converted according to known ATP concentrations, perfectly reconstruct IC50 values determined by the biochemical assays. Ligand-dependent quenching of tyrosine fluorescence, together with molecular modeling and DSC-derived heats of unfolding, support the hypothesis that halogenated benzotriazoles bind in at least two alternative orientations, and those that are efficient hCK2α inhibitors bind in the orientation which TBBt adopts in its complex with maize CK2α. DSC-derived apparent heat for ligand binding (ΔΔHbind) is driven by intermolecular electrostatic interactions between Lys68 and the triazole ring of the ligand, as indicated by a good correlation between ΔΔHbind and ligand pKa. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly (~40 kJ/mol), relative to possible intermolecular halogen/hydrogen bonding (less than 10 kJ/mol), in binding of halogenated benzotriazoles to the ATP-binding site of hCK2α. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Molecular mechanisms of the synergy between cysteinyl-leukotrienes and receptor tyrosine kinase growth factors on human bronchial fibroblast proliferation

    Directory of Open Access Journals (Sweden)

    Hajime Yoshisue

    2006-12-01

    Full Text Available We have reported that cysteinyl-leukotrienes (cys-LTs synergise not only with epidermal growth factor (EGF but also with platelet-derived growth factor (PDGF and fibroblast growth factor (FGF to induce mitogenesis in human bronchial fibroblasts. We now describe the molecular mechanisms underlying this synergism. Mitogenesis was assessed by incorporation of [3H]thymidine into DNA and changes in protein phosphorylation by Western blotting. Surprisingly, no CysLT receptor antagonists (MK-571, montelukast, BAY u9773 prevented the synergistic mitogenesis. LTD4 did not cause phosphorylation of EGFR nor did it augment EGF-induced phosphorylation of EGFR, and the synergy between LTD4 and EGF was not blocked by the metalloproteinase inhibitor GM6001 or by an HB-EGF neutralising antibody. The EGFR-selective kinase inhibitor, AG1478, suppressed the synergy by LTD4 and EGF, but had no effect on the synergy with PDGF and FGF. While inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C (PKC prevented the synergy, these drugs also inhibited mitogenesis elicited by EGF alone. In contrast, pertussis toxin (PTX efficiently inhibited the potentiating effect of LTD4 on EGF-induced mitogenesis, as well as that provoked by PDGF or FGF, but had no effect on mitogenesis elicited by the growth factors alone. Whereas LTD4 alone did not augment phosphorylation of extracellular signal-regulated kinase (Erk-1/2 and Akt, it increased phosphorylation of PKC in a Gi-dependent manner. Addition of LTD4 prolonged the duration of EGF-induced phosphorylation of Erk-1/2 and Akt, both of which were sensitive to PTX. The effect of cys-LTs involves a PTX-sensitive and PKC-mediated intracellular pathway leading to sustained growth factor-dependent phosphorylation of Erk-1/2 and Akt.

  9. In Vitro Inhibition of Human Sperm Creatine Kinase by Nicotine,Cotinine and Cadmium, as a Mechanism in Smoker Men Infertility

    OpenAIRE

    Mohammad Ali Ghaffari; Mohammad Abromand; Behrooz Motlagh

    2009-01-01

    Background: Nicotine, cotinine and cadmium are harmful components of cigarettes that have aneffect on human reproductive function. Although the effects of cigarette smoke on male reproductivefunction is characterized in several articles its mechanism of action is still unknown.In the present study, we investigate the effect of nicotine, cotinine and cadmium on human spermcreatine kinase activity in vitro.Materials and Methods: Total creatine kinase activity is measured in sperm homogenates af...

  10. The production of VEGF involving MAP kinase activation by low level laser therapy in human granulosa cells

    Science.gov (United States)

    Utsunomiya-Kai, Yufuko; Kai, Kentaro; Miyakawa, Isao; Ohshiro, Toshio; Narahara, Hisashi

    2012-01-01

    Objective: The function of granulosa cells is regulated by various hormones and growth factors. Our aim is to clarify the regulation of vascular endothelial growth factor (VEGF) production via mitogen-activated protein kinase (MAPK) induced by low level laser therapy (LLLT) in human granulosa cells. Methods: A human granulosa cell line, KGN cells, were cultured and incubated after LLLT (60mW, GaAlAs 830nm). The levels of VEGF in the culture media were determined by an enzyme-linked immunosorbent assay. The activation of MAP kinase in KGN cells was detected by western blot analysis. Results: VEGF production was significantly increased by LLLT in a time-dependent manner. MAP kinase activity was increased by LLLT. In addition it was enhanced by LLLT and follicle-stimulating hormone (FSH) stimulation. Conclusions: The results suggested that VEGF is induced by LLLT through mechanisms involving MAPK. The increase in VEGF may contribute to neovascularization, which in turn would promote various ovulation phenomena as well as follicular growth. PMID:24511196

  11. Significance of Ser-188 in human mitochondrial NAD kinase as determined by phosphomimetic and phosphoresistant amino-acid substitutions.

    Science.gov (United States)

    Kawabata, Yutaka; Murata, Kousaku; Kawai, Shigeyuki

    2015-12-25

    Human mitochondrial NAD kinase is a crucial enzyme responsible for the synthesis of mitochondrial NADP(+). Despite its significance, little is known about the regulation of this enzyme in the mitochondria. Several putative and known phosphorylation sites within the protein have been found using phosphoproteomics, and here, we examined the effect of phosphomimetic mutations at six of these sites. The enzymatic activity was downregulated by a substitution of an Asp residue at Ser-289 and Ser-376, but not a substitution of Ala, suggesting that the phosphorylation of these residues downregulates the enzyme. Moreover, the activity was completely inhibited by substituting Ser-188 with an Asp, Glu, or in particular Ala, which highlights two possibilities: first, that Ser-188 is critical for catalytic activity, and second, that phosphorylation of Ser-188 inhibits the activity. Ser-188, Ser-289, and Ser-376 were found to be highly conserved in the primary structures of mitochondrial NAD kinase homologs in higher animals. Moreover, Ser-188 has been frequently detected in human and mouse phosphorylation site studies, whereas Ser-289 and Ser-376 have not. Taken together, this indicates that Ser-188 (and perhaps the other residues) is an important phosphorylation site that can downregulate the NAD kinase activity of this critical enzyme. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Protein kinase inhibitors CK59 and CID755673 alter primary human NK cell effector functions

    Directory of Open Access Journals (Sweden)

    Maxi eScheiter

    2013-03-01

    Full Text Available Natural killer (NK cells are part of the innate immune response and play a crucial role in the defense against tumors and virus-infected cells. Their effector functions include the specific killing of target cells, as well as the modulation of other immune cells by cytokine release. Kinases constitute a relevant part in signaling, are prime targets in drug research and the protein kinase inhibitor Dasatinib is already used for immune-modulatory theraphies. In this study, we have tested the effects of the kinase inhibitors CK59 and CID755673. These inhibitors are directed against CaMKII (CK59 and PKD family kinases (CID755673 that were previously suggested as novel components of NK activation pathways. Here, we use a multi-parameter, FACS-based assay to validate the influence of CK59 and CID755673 on the effector functions of primary NK cells. Dose dependent treatment with CK59 and CID755673 indeed results in a significant reduction of NK cell degranulation markers and cytokine release in freshly isolated PBMC populations from healthy blood donors. These results underline the importance of CaMKII for NK cell signaling and suggest PKD2 as a novel signaling component in NK cell activation. Notably, kinase inhibition studies on pure NK cell populations indicate significant donor variations.

  13. Induction of Macrophage Function in Human THP-1 Cells Is Associated with Rewiring of MAPK Signaling and Activation of MAP3K7 (TAK1) Protein Kinase.

    Science.gov (United States)

    Richter, Erik; Ventz, Katharina; Harms, Manuela; Mostertz, Jörg; Hochgräfe, Falko

    2016-01-01

    Macrophages represent the primary human host response to pathogen infection and link the immediate defense to the adaptive immune system. Mature tissue macrophages convert from circulating monocyte precursor cells by terminal differentiation in a process that is not fully understood. Here, we analyzed the protein kinases of the human monocytic cell line THP-1 before and after induction of macrophage differentiation by using kinomics and phosphoproteomics. When comparing the macrophage-like state with the monocytic precursor, 50% of the kinome was altered in expression and even 71% of covered kinase phosphorylation sites were affected. Kinome rearrangements are for example characterized by a shift of overrepresented cyclin-dependent kinases associated with cell cycle control in monocytes to calmodulin-dependent kinases and kinases involved in proinflammatory signaling. Eventually, we show that monocyte-to-macrophage differentiation is associated with major rewiring of mitogen-activated protein kinase signaling networks and demonstrate that protein kinase MAP3K7 (TAK1) acts as the key signaling hub in bacterial killing, chemokine production and differentiation. Our study proves the fundamental role of protein kinases and cellular signaling as major drivers of macrophage differentiation and function. The finding that MAP3K7 is central to macrophage function suggests MAP3K7 and its networking partners as promising targets in host-directed therapy for macrophage-associated disease.

  14. Induction of Macrophage Function in Human THP-1 Cells is Associated with MAPK Signaling and Activation of MAP3K7 (TAK1 Protein Kinase

    Directory of Open Access Journals (Sweden)

    Erik eRichter

    2016-03-01

    Full Text Available Macrophages represent the primary human host response to pathogen infection and link the immediate defense to the adaptive immune system. Mature tissue macrophages convert from circulating monocyte precursor cells by terminal differentiation in a process that is not fully understood. Here, we analyzed the protein kinases of the human monocytic cell line THP-1 before and after induction of macrophage differentiation by using kinomics and phosphoproteomics. When comparing the macrophage-like state with the monocytic precursor, 50% of the kinome was altered in expression and even 71% of covered kinase phosphorylation sites were affected. Kinome rearrangements are for example characterized by a shift of overrepresented cycline-dependent kinases associated with cell cycle control in monocytes to calmodulin-dependent kinases and kinases involved in proinflammatory signaling. Eventually, we show that monocyte-to-macrophage differentiation is associated with major rewiring of mitogen-activated protein kinase signaling networks and demonstrate that protein kinase MAP3K7 (TAK1 acts as the key signaling hub in bacterial killing, chemokine production and differentiation. Our study proves the fundamental role of protein kinases and cellular signaling as major drivers of macrophage differentiation and function. The finding that MAP3K7 is central to macrophage function suggests MAP3K7 and its networking partners as promising targets in host-directed therapy for macrophage-associated disease.

  15. The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    DEFF Research Database (Denmark)

    Hendus-Altenburger, Ruth; Pedraz Cuesta, Elena; Olesen, Christina Wilkens

    2016-01-01

    BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify...... the human Na(+)/H(+) exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D...... and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2....

  16. Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Gomez, M.L.; Tellez-Inon, M.T. (Instituto de Ingenieria Genetica y Biologia Molecular, Buenos Aires (Argentina)); Medrano, E.E.; Cafferatta, E.G.A. (Instituto de Investigaciones Bioquimicas Fundacion Campomar, Buenos Aires (Argentina))

    1988-03-01

    The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

  17. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesothelioma cells: role of mitogen-activated protein kinase pathway.

    Science.gov (United States)

    Trombino, Sonya; Cesario, Alfredo; Margaritora, Stefano; Granone, PierLuigi; Motta, Giovanni; Falugi, Carla; Russo, Patrizia

    2004-01-01

    This study presents data suggesting that both human mesothelioma (cell lines and human mesothelioma biopsies) and human normal mesothelial cells express receptors for acetylcholine and that stimulation of these receptors by nicotine prompted cell growth via activation of nicotinic cholinergic receptors. Thus, these data demonstrate that: (a) human mesothelioma cells and human biopsies of mesothelioma as well as of normal pleural mesothelial cells express functionally alpha-7 nicotinic acethlycholine receptors, evaluated by alpha-bungarotoxin-FITC binding, receptor binding assay, Western blot, and reverse transcription-PCR; (b) choline acetyltransferase immunostaining is present in mesothelioma cells; (c) mesothelioma cell growth is modulated by the cholinergic system in which agonists (i.e., nicotine) has a proliferative effect, and antagonists (i.e., curare) has an inhibitory effect, evaluated by cell cloning, DNA synthesis and cell cycle; (d) nicotine induces Ca(+2) influx, evaluated by [(45)Ca(2+)] uptake, and consequently activation of mitogen-activated protein kinase pathway (extracellular signal-regulated kinase and p90(RSK) phosphorylation), evaluated by Western blot; and (e) apoptosis mechanisms in mesothelioma cells are under the control of the cholinergic system (nicotine antiapoptotic via induction of nuclear factor-kappaB complexes and phosphorylation of Bad at Ser(112); curare proapoptotic via G(0)-G(1) arrest p21(waf-1) dependent but p53 independent). The involvement of the nonneuronal cholinergic system in mesothelioma appears reasonable and open up new therapeutic strategies.

  18. Short interfering RNA inhibits Rift Valley fever virus replication and degradation of protein kinase R in human cells

    Directory of Open Access Journals (Sweden)

    Bonto Faburay

    2016-11-01

    Full Text Available Rift Valley fever virus (RVFV is a mosquito-borne zoonotic pathogen causing severe outbreaks in humans and livestock in sub-Saharan Africa and the Arabian Peninsula. Human infections are characterized by fever, sometimes leading to encephalitis, retinitis, hemorrhagic fever and occasionally death. There are currently no fully licensed vaccines or effective therapies for human use. Gene silencing mediated by double-stranded short interfering RNA (siRNA is a sequence-specific, highly conserved mechanism in eukaryotes, which serves as an antiviral defence mechanism. Here, we demonstrate that siRNA duplexes directed against the RVFV nucleoprotein can effectively inhibit RVFV replication in human (MRC5 cells and African green monkey cells (Vero E6 cells. Using these cells, we demonstrate that individual or complex siRNAs, targeting the RVFV nucleoprotein gene completely abrogate viral protein expression and prevent degradation of the host innate antiviral factor, protein kinase R (PKR. Importantly, pre-treatment of cells with the nucleoprotein-specific siRNAs markedly reduces the virus titer. The antiviral effect of the siRNAs was not attributable to interferon or the interferon response effector molecule, protein kinase R. Thus, the antiviral activity of RVFV nucleoprotein-specific siRNAs may provide novel therapeutic strategy against RVFV infections in animals and humans.

  19. Human pre-B cell receptor signal transduction: evidence for distinct roles of PI3kinase and MAP-kinase signalling pathways

    Science.gov (United States)

    Anbazhagan, Kolandaswamy; Rabbind Singh, Amrathlal; Isabelle, Piec; Stella, Ibata; Céline, Alleaume-De Martel; Bissac, Eliane; Bertrand, Brassart; Rémy, Nyga; Naomi, Taylor; Vincent, Fuentes; Rochette, Jacques; Lassoued, Kaïss

    2013-01-01

    Pre-BCR acts as a critical checkpoint in B cell development. However, its signalling cascade still remains indistinctly characterised in human. We investigated pre-BCR signalling pathway to examine its regulation in normal primary pre-B lymphocytes and pre-B cell lines. In cell lines, early signalling events occurring after pre-BCR stimulation include phosphorylation of Lyn, Blk and Syk together with ZAP70, Btk, Vav, PLC-γ2 and various adaptor proteins, such as BLNK, LAB, LAT and SLP-76. Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation. PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways. Immediate nuclear export of FoxO3A and delayed import of IRF4 were additional events observed after pre-BCR crosslinking in primary cells. Pre-BCR-induced down-regulation of Rag1, Rag2, E2A and Pax5 transcripts occurred in a PI3K-dependent manner. Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal. PMID:25400915

  20. Differentially activated Src kinase in chemo-naïve human primary osteosarcoma cells and effects of a Src kinase inhibitor.

    Science.gov (United States)

    Laschi, Marcella; Bernardini, Giulia; Geminiani, Michela; Manetti, Fabrizio; Mori, Mattia; Spreafico, Adriano; Campanacci, Domenico; Capanna, Rodolfo; Schenone, Silvia; Botta, Maurizio; Santucci, Annalisa

    2017-11-01

    The therapeutic treatment of osteosarcoma (OS), a rare malignant teenage cancer of the skeletal system, still represents a great challenge as patient survival after conventional protocol chemotherapy treatment has not improved in the last four decades leaving poor patient prognoses. Therefore, many efforts have been done to find increasingly reliable OS cell models and to identify "druggable" targets in OS, in order to identify novel effective therapeutic approaches and treatment strategies. In this contest, the more successful use of patient-derived cell cultures in respect to human commercial lines and findings of Src kinase deregulation in cancer, prompted us to study for the first time the activation state of Src and the potential activity of our Src inhibitor SI-83 in a number of chemo-naïve patient-derived primary OS cells. We here demonstrate that Src is hyperactivated in OS cells in respect to the nonmalignant counterpart and that SI-83 is able to strongly decrease cell viability, proliferation, Src416 phosphorylation, and cell migration. © 2017 BioFactors, 43(6):801-811, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  1. Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

    Science.gov (United States)

    Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

    2016-09-01

    Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  2. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    Energy Technology Data Exchange (ETDEWEB)

    Manceur, Aziza P. [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Tseng, Michael [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Holowacz, Tamara [Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Witterick, Ian [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Department of Otolaryngology, Head and Neck Surgery, University of Toronto, ON (Canada); Weksberg, Rosanna [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); McCurdy, Richard D. [The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); Warsh, Jerry J. [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Audet, Julie, E-mail: julie.audet@utoronto.ca [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada)

    2011-09-10

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  3. Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon.

    Directory of Open Access Journals (Sweden)

    Claire Marie Filone

    2010-11-01

    Full Text Available As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.

  4. Characterization of casein kinase II in human colonic carcinomas after heterotransplantation into nude mice

    DEFF Research Database (Denmark)

    Seitz, G; Münstermann, U; Schneider, H R

    1989-01-01

    Casein kinase II (CKII) activity in colorectal tumours was compared before and after heterotransplantation onto nude mice. The test revealed that the enzyme activity was about two-fold enhanced in the tumours isolated from the nude mice when compared to the respective primary tumours from which...

  5. Resistance artery creatine kinase mRNA and blood pressure in humans

    NARCIS (Netherlands)

    Karamat, Fares A.; Oudman, Inge; Ris-Stalpers, Carrie; Afink, Gijs B.; Keijser, Remco; Clark, Joseph F.; van Montfrans, Gert A.; Brewster, Lizzy M.

    2014-01-01

    Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of

  6. Regulation of serotonin transport in human platelets by tyrosine kinase Syk.

    Science.gov (United States)

    Pavanetto, Martina; Zarpellon, Alessandro; Borgo, Christian; Donella-Deana, Arianna; Deana, Renzo

    2011-01-01

    Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the regulation of numerous neuro-physiological processes. The circulating level of 5-HT is regulated by the membrane transporter SERT present both in the presynaptic nerve terminals and blood platelets. 5-HT transport is a process tightly regulated by a variety of factors including protein phosphorylation. Aim of this study was to ascertain if also the SERT Tyr-phosphorylation mediated by Syk-kinase concurs to the regulation of SERT activity. Indeed we found that 5-HT uptake decreased upon platelet exposure to piceatannol or Syk-inhibitor II, two structurally unrelated inhibitors of the tyrosine-kinase Syk. Tyr-phosphorylation of anti-SERT-immuno-stained proteins in membrane extracts and in anti-SERT-immuno-precipitates, decreased upon platelet treatment with piceatannol, in parallel with a reduction of Syk-activity. Syk was immuno-revealed in the anti-SERT immuno-precipitates, which displayed a piceatannol-sensitive kinase activity towards SERT itself and the Syk-substrate α-sinuclein. Syk inhibitors also caused a decrease of the monensin-induced 5-HT-efflux from platelets and of imipramine binding to them. It is concluded that, in addition to the phosphorylation of SERT mediated by various other kinases, also that catalyzed by Syk might play an important role in the 5-HT transport, likely favoring the transporter conformation exposing the neurotransmitter binding sites. Copyright © 2011 S. Karger AG, Basel.

  7. Effect of Quercetin flavonoid on structural changes of recombinant human FGFR2b kinase domain

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    Benyamin Alimohammadi

    2017-08-01

    Conclusion: The tertiary structural change of FGFR2b kinase domain represents a conformational change that may have a critical role in signal transduction cascade. Thus, this molecular transduction inconsistency can lead to cellular transduction complication; and as a result, inhibit the development and multiplication of  cancerous cells.

  8. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Chen-Shuang Li

    2016-01-01

    Full Text Available Human umbilical cord mesenchymal stem cells (hUCMSCs are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering.

  9. Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

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    Jing Zhang

    2017-12-01

    Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

  10. Cloning and quantitative determination of the human Ca2+/calmodulin-dependent protein kinase II (CaMK II) isoforms in human beta cells.

    Science.gov (United States)

    Rochlitz, H; Voigt, A; Lankat-Buttgereit, B; Göke, B; Heimberg, H; Nauck, M A; Schiemann, U; Schatz, H; Pfeiffer, A F

    2000-04-01

    The Ca2+/calmodulin-dependent protein kinase II (CaMK II) is highly expressed in pancreatic islets and associated with insulin secretion vesicles. The suppression of CaMK II disturbs insulin secretion and insulin gene expression. There are four isoforms of CaMK II, alpha to delta, that are expressed from different genes in mammals. Our aim was to identify the isoforms of CaMK II expressed in human beta cells by molecular cloning from a human insulinoma cDNA library and to assess its distribution in humans. The previously unknown complete coding sequences of human CaMK IIbeta and the kinase domain of CaMK IIdelta were cloned from a human insulinoma cDNA library. Quantitative determination of CaMK II isoform mRNA was carried out in several tissues and beta cells purified by fluorescence activated cell sorting and compared to the housekeeping enzyme pyruvate dehydrogenase. We found CaMK IIbeta occurred in three splice variants and was highly expressed in endocrine tissues such as adrenals, pituitary and beta cells. Liver showed moderate expression but adipose tissue or lymphocytes had very low levels of CaMK IIbeta-mRNA. In human beta cells CaMK IIbeta and delta were expressed equally with pyruvate dehydrogenase whereas tenfold lower expression of CaMK IIgamma and no expression of CaMK IIalpha were found. Although CaMK IIdelta is ubiquitously expressed, CaMK IIbeta shows preferential expression in neuroendocrine tissues. In comparison with the expression of a key regulatory enzyme in glucose oxidation, pyruvate dehydrogenase, two of the four CaM kinases investigated are expressed at equally high levels, which supports an important role in beta-cell physiology. These results provide the basis for exploring the pathophysiological relevance of CaMK IIbeta in human diabetes.

  11. Histamine induces activation of protein kinase D that mediates tissue factor expression and activity in human aortic smooth muscle cells

    Science.gov (United States)

    Hao, Feng; Wu, Daniel Dongwei; Xu, Xuemin

    2012-01-01

    Histamine, an inflammatory mediator, has been shown to influence the pathogenesis of vascular wall cells. However, the molecular basis of its influence is not well understood. Our data reveal that histamine markedly induces protein kinase D (PKD) activation in human aortic smooth muscle cells. PKD belongs to a family of serine/threonine protein kinases, and its function in vascular disease is largely unknown. Our data show that histamine-induced PKD phosphorylation is dependent on the activation of histamine receptor 1 and protein kinase C (PKC). To determine the role of PKD in the histamine pathway, we employed a small-interfering RNA approach to downregulate PKD expression and found that PKD1 and PKD2 are key mediators for expression of tissue factor (TF), which is the key initiator of blood coagulation and is important for thrombosis. Our results show that PKD2 predominantly mediates histamine-induced TF expression via the p38 mitogen-activated protein kinase (MAPK) pathway, whereas PKD1 mediates histamine-induced TF expression through a p38 MAPK-independent pathway. We demonstrate that histamine induces TF expression via the PKC-dependent PKD activation. Our data provide the first evidence that PKD is a new component in histamine signaling in live cells and that PKD has a novel function in the histamine signaling pathway leading to gene expression, as evidenced by TF expression. Importantly, our data reveal a regulatory link from histamine to PKD and TF, providing new insights into the mechanisms of coagulation and the development of atherothrombosis. PMID:23001835

  12. Tissue injury after lithium treatment in human and rat postnatal kidney involves glycogen synthase kinase 3β-positive epithelium

    DEFF Research Database (Denmark)

    Kjaersgaard, Gitte; Madsen, Kirsten; Marcussen, Niels

    2012-01-01

    It was hypothesized that lithium causes accelerated and permanent injury to the postnatally developing kidney through entry into epithelial cells of the distal nephron and inhibition of glycogen synthase kinase-3β (GSK-3β). GSK-3β immunoreactivity was associated with glomeruli, thick ascending limb...... of Henle's loop and collecting ducts in developing and adult human and rat kidney. In rats, the abundance of inactive, phosphorylated GSK-3β (pGSK-3β) protein decreased during postnatal development. After feeding dams with litters lithium (50 mmol Li/kg chow, postnatal (P) day 7-28), the offspring showed...

  13. New applications for known drugs: Human glycogen synthase kinase 3 inhibitors as modulators of Aspergillus fumigatus growth.

    Science.gov (United States)

    Sebastián-Pérez, Víctor; Manoli, Maria-Tsampika; Pérez, Daniel I; Gil, Carmen; Mellado, Emilia; Martínez, Ana; Espeso, Eduardo A; Campillo, Nuria E

    2016-06-30

    Invasive aspergillosis (IA) is one of the most severe forms of fungi infection. IA disease is mainly due to Aspergillus fumigatus, an air-borne opportunistic pathogen. Mortality rate caused by IA is still very high (50-95%), because of difficulty in early diagnostics and reduced antifungal treatment options, thus new and efficient drugs are necessary. The aim of this work is, using Aspergillus nidulans as non-pathogen model, to develop efficient drugs to treat IA. The recent discovered role of glycogen synthase kinase-3 homologue, GskA, in A. fumigatus human infection and our previous experience on human GSK-3 inhibitors focus our attention on this kinase as a target for the development of antifungal drugs. With the aim to identify effective inhibitors of colonial growth of A. fumigatus we use A. nidulans as an accurate model for in vivo and in silico studies. Several well-known human GSK-3β inhibitors were tested for inhibition of A. nidulans colony growth. Computational tools as docking studies and binding site prediction was used to explain the different biological profile of the tested inhibitors. Three of the five tested hGSK3β inhibitors are able to reduce completely the colonial growth by covalent bind to the enzyme. Therefore these compounds may be useful in different applications to eradicate IA. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  14. Evaluation of Fluorescent Analogs of Deoxycytidine for Monitoring DNA Transitions from Duplex to Functional Structures

    Directory of Open Access Journals (Sweden)

    Yogini P. Bhavsar

    2011-01-01

    Full Text Available Topological variants of single-strand DNA (ssDNA structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC and 1,3-diaza-2-oxophenoxazine (tC∘, can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC∘ is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions.

  15. Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5.

    Science.gov (United States)

    Bickel, Kathleen G; Mann, Barbara J; Waitzman, Joshua S; Poor, Taylor A; Rice, Sarah E; Wadsworth, Patricia

    2017-09-01

    Spindle formation in mammalian cells requires precise spatial and temporal regulation of the kinesin-5, Eg5, which generates outward force to establish spindle bipolarity. Our results demonstrate that Eg5 is phosphorylated in cultured cells by Src family kinases (SFKs) at three sites in the motor head: Y125, Y211, and Y231. Mutation of these sites diminishes motor activity in vitro, and replacement of endogenous Eg5 with phosphomimetic Y211 in LLC-Pk1 cells results in monopolar spindles, consistent with loss of Eg5 activity. Cells treated with SFK inhibitors show defects in spindle formation, similar to those in cells expressing the nonphosphorylatable Y211 mutant, and distinct from inhibition of other mitotic kinases. We propose that this phosphoregulatory mechanism tunes Eg5 enzymatic activity for optimal spindle morphology. © 2017 Wiley Periodicals, Inc.

  16. Gastrin induces sodium-hydrogen exchanger 3 phosphorylation and mTOR activation via a phosphoinositide 3-kinase-/protein kinase C-dependent but AKT-independent pathway in renal proximal tubule cells derived from a normotensive male human.

    Science.gov (United States)

    Liu, Tianbing; Jose, Pedro A

    2013-02-01

    Gastrin is natriuretic, but its renal molecular targets and signal transduction pathways are not fully known. In this study, we confirmed the existence of CCKBR (a gastrin receptor) in male human renal proximal tubule cells and discovered that gastrin induced S6 phosphorylation, a downstream component of the phosphatidylinositol 3 kinase (PI3 kinase)-mammalian target of rapamycin pathway. Gastrin also increased the phosphorylation of sodium-hydrogen exchanger 3 (NHE3) at serine 552, caused its internalization, and decreased its expression at the cell surface and NHE activity. The phosphorylation of NHE3 and S6 was dependent on PI3 kinases because it was blocked by 2 different PI3-kinase inhibitors, wortmannin and LY294,002. The phosphorylation of NHE3 and S6 was not affected by the protein kinase A inhibitor H-89 but was blocked by a pan-PKC (chelerythrine) and a conventional PKC (cPKC) inhibitor (Gö6976) (10 μM) and an intracellular calcium chelator, 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The cPKC involved was probably PKCα because it was phosphorylated by gastrin. The gastrin-mediated phosphorylation of NHE3, S6, and PKCα was via phospholipase C because it was blocked by a phospholipase C inhibitor, U73122 (10 μM). The phosphorylation (activation) of AKT, which is usually upstream of mammalian target of rapamycin in the classic PI3 kinase-AKT-p70S6K signaling pathway, was not affected, suggesting that the gastrin-induced phosphorylation of NHE3 and S6 is dependent on both PI3 kinase and PKCα but not AKT.

  17. Short waves-induced enhancement of proliferation of human chondrocytes: involvement of extracellular signal-regulated map-kinase (erk).

    Science.gov (United States)

    Wang, Jue-Long; Chan, Rai-Chi; Cheng, He-Hsiung; Huang, Chun-Jen; Lu, Yih-Chau; Chen, I-Shu; Liu, Shiuh-Inn; Hsu, Shu-Shong; Chang, Hong-Tai; Huang, Jong-Khing; Chen, Jin-Shyr; Ho, Chin-Man; Jan, Chung-Ren

    2007-07-01

    1. Short-wave diathermy (SWD) is a form of radiofrequency radiation that is used therapeutically by physiotherapists. The cellular mechanisms of SWD are unclear. The present study was performed to explore the effect of different conditions of short-wave exposure on the proliferation of cultured human chondrocytes. 2. Cells exposed to short waves once per day for seven consecutive days exhibited a significant increase in proliferation by 42% compared with the control cells. In cells that were treated with short waves twice per day for seven consecutive days, or only once on Day 1 and then examined for proliferation on Day 7, cell proliferation was greater than the control cells by 40% and 30%, respectively. 3. Given the importance of mitogen-activated protein kinases (MAPK) in the proliferation of different cell types, efforts were extended to explore the role of three major types of MAPK; that is, extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal protein kinase (JNK) and p38. 4. It was found that the level of phosphorylated ERK (phospho-ERK 1 and ERK 2) increased significantly within 5-120 min following consecutive exposure to short waves for 7 days. Exposure to short waves failed to alter the intensity of phosphorylated JNK and p38 within 0-240 min. 5. Cells were exposed to short waves once for seven consecutive days in the presence of 0, 10 micromol/L, 20 micromol/L or 50 micromol/L PD98059 (an ERK inhibitor). PD98059 totally inhibited short waves-induced enhancement of proliferation without altering normal control viability. In the presence of short waves and PD98059, the cell viability was lower than the normal control. Together, the data suggest that short waves could increase proliferation in human chondrocytes through activation of the ERK pathway, which is also involved in maintaining normal cell proliferation under physiological conditions.

  18. Elevated cyclin A associated kinase activity promotes sensitivity of metastatic human cancer cells to DNA antimetabolite drug.

    Science.gov (United States)

    Wang, Jin; Yin, Hailin; Panandikar, Ashwini; Gandhi, Varsha; Sen, Subrata

    2015-08-01

    Drug resistance is a major obstacle in successful systemic therapy of metastatic cancer. We analyzed the involvement of cell cycle regulatory proteins in eliciting response to N (phosphonoacetyl)-L-aspartate (PALA), an inhibitor of de novo pyrimidine synthesis, in two metastatic variants of human cancer cell line MDA-MB-435 isolated from lung (L-2) and brain (Br-1) in nude mouse, respectively. L-2 and Br-l cells markedly differed in their sensitivity to PALA. While both cell types displayed an initial S phase delay/arrest, Br-l cells proliferated but most L-2 cells underwent apoptosis. There was distinct elevation in cyclin A, and phosphorylated Rb proteins concomitant with decreased expression of bcl-2 protein in the PALA treated L-2 cells undergoing apoptosis. Markedly elevated cyclin A associated and cdk2 kinase activities together with increased E2F1-DNA binding were detected in these L-2 cells. Induced ectopic cyclin A expression sensitized Br-l cells to PALA by activating an apoptotic pathway. Our findings demonstrate that elevated expression of cyclin A and associated kinase can activate an apoptotic pathway in cells exposed to DNA antimetabolites. Abrogation of this pathway can lead to resistance against these drugs in metastatic variants of human carcinoma cells.

  19. Differential role of human choline kinase alpha and beta enzymes in lipid metabolism: implications in cancer onset and treatment.

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    David Gallego-Ortega

    Full Text Available BACKGROUND: The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK is the first enzyme of the Kennedy branch of synthesis of phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKalpha and ChoKbeta isoforms, the first one with two different variants of splicing. Recently ChoKalpha has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKbeta in carcinogenesis has been reported. METHODOLOGY/PRINCIPAL FINDINGS: Here we compare the in vitro and in vivo properties of ChoKalpha1 and ChoKbeta in lipid metabolism, and their potential role in carcinogenesis. Both ChoKalpha1 and ChoKbeta showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKbeta display an ethanolamine kinase role, ChoKalpha1 present a dual choline/ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKalpha1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKbeta overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKalpha1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKbeta mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKalpha1 than ChoKbeta. CONCLUSION/SIGNIFICANCE: This study represents the first evidence of the distinct metabolic role of ChoKalpha and ChoKbeta isoforms, suggesting different physiological roles and implications in human

  20. AMP-activated protein kinase activation mediates CCL3-induced cell migration and matrix metalloproteinase-2 expression in human chondrosarcoma

    Science.gov (United States)

    2013-01-01

    Chemokine (C-C motif) ligand 3 (CCL3), also known as macrophage inflammatory protein-1α, is a cytokine involved in inflammation and activation of polymorphonuclear leukocytes. CCL3 has been detected in infiltrating cells and tumor cells. Chondrosarcoma is a highly malignant tumor that causes distant metastasis. However, the effect of CCL3 on human chondrosarcoma metastasis is still unknown. Here, we found that CCL3 increased cellular migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. Pre-treatment of cells with the MMP-2 inhibitor or transfection with MMP-2 specific siRNA abolished CCL3-induced cell migration. CCL3 has been reported to exert its effects through activation of its specific receptor, CC chemokine receptor 5 (CCR5). The CCR5 and AMP-activated protein kinase (AMPK) inhibitor or siRNA also attenuated CCL3-upregulated cell motility and MMP-2 expression. CCL3-induced expression of MMP-2 and migration were also inhibited by specific inhibitors, and inactive mutants of AMPK, p38 mitogen activated protein kinase (p38 or p38-MAPK), and nuclear factor κB (NF-κB) cascades. On the other hand, CCL3 treatment demonstrably activated AMPK, p38, and NF-κB signaling pathways. Furthermore, the expression levels of CCL3, CCR5, and MMP-2 were correlated in human chondrosarcoma specimens. Taken together, our results indicate that CCL3 enhances the migratory ability of human chondrosarcoma cells by increasing MMP-2 expression via the CCR5, AMPK, p38, and NF-κB pathways. PMID:24047437

  1. Pharmacological Inhibition of Protein Kinase G1 Enhances Bone Formation by Human Skeletal Stem Cells Through Activation of RhoA-Akt Signaling

    DEFF Research Database (Denmark)

    Kermani, Abbas Jafari; Siersbaek, Majken S; Chen, Li

    2015-01-01

    Development of novel approaches to enhance bone regeneration is needed for efficient treatment of bone defects. Protein kinases play a key role in regulation of intracellular signal transduction pathways, and pharmacological targeting of protein kinases has led to development of novel treatments...... for several malignant and nonmalignant conditions. We screened a library of kinase inhibitors to identify small molecules that enhance bone formation by human skeletal (stromal or mesenchymal) stem cells (hMSC). We identified H-8 (known to inhibit protein kinases A, C, and G) as a potent enhancer of ex vivo...... that pharmacological inhibition of PRKG1 in hMSC implanted at the site of bone defect can enhance bone regeneration. Stem Cells 2015....

  2. Ca(2+)/calmodulin-dependent protein kinase IIβ and IIδ mediate TGFβ-induced transduction of fibronectin and collagen in human pulmonary fibroblasts.

    Science.gov (United States)

    Mukherjee, Subhendu; Sheng, Wei; Sun, Rui; Janssen, Luke J

    2017-04-01

    It is now clear that in addition to activating several complex kinase pathways (Smad, MAP kinase, PI3 kinase), TGFβ also acts by elevating cytosolic Ca(2+) concentration within human pulmonary fibroblasts. Ca(2+)/calmodulin-dependent protein kinase II (CamK II) is also known to regulate gene expression in fibroblasts. In this study, we examined the interactions between calcium signaling, activation of CamK and other kinases, and extracellular matrix (ECM) gene expression. Human pulmonary fibroblasts were cultured and stimulated with artificially generated Ca(2+) pulses in the absence of TGFβ, or with TGFβ (1 nM) or vehicle in the presence of various blockers of Ca(2+) signaling. PCR and Western blotting were used to measure gene expression and protein levels, respectively. We found that Ca(2+) pulses in the absence of TGFβ increased ECM gene expression in a pulse frequency-dependent manner, and that blocking Ca(2+) signaling and the CamK II pathway significantly reduced TGFβ-mediated ECM gene expression, without having any effects on other kinase pathways (Smad, PI3 kinase, or MAP kinase). We also found that TGFβ elevated the expression of CamK IIβ and CamK IIδ, while siRNA silencing of those two subtypes significantly reduced TGFβ-mediated expression of collagen A1 and fibronectin 1. Our data suggest that TGFβ induces the expression of CamK IIβ and CamK IIδ, which in turn are activated by TGFβ-evoked Ca(2+) waves in a frequency-dependent manner, leading to increased expression of ECM proteins. Copyright © 2017 the American Physiological Society.

  3. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Clausen, Anders R.; Girandon, Lenart; Ali, Ashfaq

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5′ of a deoxyribonucleoside. This salvage pathway is well characterized....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.......7.1.21)‐like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T‐DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a...

  4. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Anders Ranegaard Clausen, Anders Ranegaard; Girandon, Lenart; Ali, Ashfaq

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.......7.1.21)-like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a...

  5. Triptolide-Mediated Apoptosis by Suppression of Focal Adhesion Kinase through Extrinsic and Intrinsic Pathways in Human Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Haw-Young Kwon

    2013-01-01

    Full Text Available Triptolide (TPL has been shown to inhibit cell proliferation and induce apoptosis in various human cancer cells; however, the precise mechanism of apoptosis induced by TPL in human melanoma cells has not yet been elucidated. In this study, we investigated the precise mechanism underlying cytocidal effects of TPL on human melanoma cells. Treatment of human melanoma cells with TPL significantly inhibited cell growth and induced apoptosis, as evidenced by flow cytometry and annexin V-fluorescein isothiocyanate analyses. TPL increased the levels of Fas and Fas-associated death domain (FADD and induced cleavage of Bid by activation of caspase-8 and cytochrome c release from mitochondria to the cytosol, which resulted in activation of caspase-9 and caspase-3. Moreover, TPL-induced apoptosis in SK-MEL-2 cells was mediated through dephosphorylation of focal adhesion kinase (FAK and its cleavage by caspase-8-mediated caspase-3 activation via upregulation of Fas expression. We also found that TPL mediated the dissociation of receptor-interacting protein (RIP from FAK and enhanced the formation of RIP/Fas complex formation initiating cell death. In conclusion, our data firstly demonstrated that TPL induces apoptosis by both extrinsic and intrinsic apoptosis pathways in human melanoma cells and identified that RIP shuttles between Fas and FAK to mediate apoptosis.

  6. Universal quantitative kinase assay based on diagonal SCX chromatography and stable isotope dimethyl labeling provides high-definition kinase consensus motifs for PKA and human Mps1

    NARCIS (Netherlands)

    Hennrich, M.L.|info:eu-repo/dai/nl/314406778; Marino, F.|info:eu-repo/dai/nl/339461799; Groenewold, V.; Kops, G.J.P.L.; Mohammed, S.|info:eu-repo/dai/nl/30483632X; Heck, A.J.R.|info:eu-repo/dai/nl/105189332

    2013-01-01

    In order to understand cellular signaling, a clear understanding of kinase–substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method

  7. The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes.

    Science.gov (United States)

    Witte, Katrin; Koch, Egon; Volk, Hans-Dieter; Wolk, Kerstin; Sabat, Robert

    2015-01-01

    Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells.

  8. Isomeric mono-, di-, and tri-bromobenzo-1H-triazoles as inhibitors of human protein kinase CK2α.

    Directory of Open Access Journals (Sweden)

    Romualda Wąsik

    Full Text Available To further clarify the role of the individual bromine atoms of 4,5,6,7-tetrabromotriazole (TBBt, a relatively selective inhibitor of protein kinase CK2, we have examined the inhibition (IC(50 of human CK2α by the two mono-, the four di-, and the two tri- bromobenzotriazoles relative to that of TBBt. Halogenation of the central vicinal C(5/C(6 atoms proved to be a key factor in enhancing inhibitory activity, in that 5,6-di-Br(2Bt and 4,5,6-Br(3Bt were almost as effective inhibitors as TBBt, notwithstanding their marked differences in pK(a for dissociation of the triazole proton. The decrease in pK(a on halogenation of the peripheral C(4/C(7 atoms virtually nullifies the gain due to hydrophobic interactions, and does not lead to a decrease in IC(50. Molecular modeling of structures of complexes of the ligands with the enzyme, as well as QSAR analysis, pointed to a balance of hydrophobic and electrostatic interactions as a discriminator of inhibitory activity. The role of halogen bonding remains debatable, as originally noted for the crystal structure of TBBt with CK2α (pdb1j91. Finally we direct attention to the promising applicability of our series of well-defined halogenated benzotriazoles to studies on inhibition of kinases other than CK2.

  9. Hyperactivation of phosphatidylinositol-3 kinase promotes escape from hormone dependence in estrogen receptor–positive human breast cancer

    Science.gov (United States)

    Miller, Todd W.; Hennessy, Bryan T.; González-Angulo, Ana M.; Fox, Emily M.; Mills, Gordon B.; Chen, Heidi; Higham, Catherine; García-Echeverría, Carlos; Shyr, Yu; Arteaga, Carlos L.

    2010-01-01

    Many breast cancers exhibit a degree of dependence on estrogen for tumor growth. Although several therapies have been developed to treat individuals with estrogen-dependent breast cancers, some tumors show de novo or acquired resistance, rendering them particularly elusive to current therapeutic strategies. Understanding the mechanisms by which these cancers develop resistance would enable the development of new and effective therapeutics. In order to determine mechanisms of escape from hormone dependence in estrogen receptor–positive (ER-positive) breast cancer, we established 4 human breast cancer cell lines after long-term estrogen deprivation (LTED). LTED cells showed variable changes in ER levels and sensitivity to 17β-estradiol. Proteomic profiling of LTED cells revealed increased phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6 kinase and p85S6 kinase as well as the PI3K substrate AKT. Inhibition of PI3K and mTOR induced LTED cell apoptosis and prevented the emergence of hormone-independent cells. Using reverse-phase protein microarrays, we identified a breast tumor protein signature of PI3K pathway activation that predicted poor outcome after adjuvant endocrine therapy in patients. Our data suggest that upon adaptation to hormone deprivation, breast cancer cells rely heavily on PI3K signaling. Our findings also imply that acquired resistance to endocrine therapy in breast cancer may be abrogated by combination therapies targeting both ER and PI3K pathways. PMID:20530877

  10. ARQ 197, a novel and selective inhibitor of the human c-Met receptor tyrosine kinase with antitumor activity.

    Science.gov (United States)

    Munshi, Neru; Jeay, Sébastien; Li, Youzhi; Chen, Chang-Rung; France, Dennis S; Ashwell, Mark A; Hill, Jason; Moussa, Magdi M; Leggett, David S; Li, Chiang J

    2010-06-01

    The met proto-oncogene is functionally linked with tumorigenesis and metastatic progression. Validation of the receptor tyrosine kinase c-Met as a selective anticancer target has awaited the emergence of selective c-Met inhibitors. Herein, we report ARQ 197 as the first non-ATP-competitive small molecule that selectively targets the c-Met receptor tyrosine kinase. Exposure to ARQ 197 resulted in the inhibition of proliferation of c-Met-expressing cancer cell lines as well as the induction of caspase-dependent apoptosis in cell lines with constitutive c-Met activity. These cellular responses to ARQ 197 were phenocopied by RNAi-mediated c-Met depletion and further demonstrated by the growth inhibition of human tumors following oral administration of ARQ 197 in multiple mouse xenograft efficacy studies. Cumulatively, these data suggest that ARQ 197, currently in phase II clinical trials, is a promising agent for targeting cancers in which c-Met-driven signaling is important for their survival and proliferation.

  11. Several Human Cyclin-Dependent Kinase Inhibitors, Structurally Related to Roscovitine, As New Anti-Malarial Agents

    Directory of Open Access Journals (Sweden)

    Sandrine Houzé

    2014-09-01

    Full Text Available In Africa, malaria kills one child each minute. It is also responsible for about one million deaths worldwide each year. Plasmodium falciparum, is the protozoan responsible for the most lethal form of the disease, with resistance developing against the available anti-malarial drugs. Among newly proposed anti-malaria targets, are the P. falciparum cyclin-dependent kinases (PfCDKs. There are involved in different stages of the protozoan growth and development but share high sequence homology with human cyclin-dependent kinases (CDKs. We previously reported the synthesis of CDKs inhibitors that are structurally-related to (R-roscovitine, a 2,6,9-trisubstituted purine, and they showed activity against neuronal diseases and cancers. In this report, we describe the synthesis and the characterization of new CDK inhibitors, active in reducing the in vitro growth of P. falciparum (3D7 and 7G8 strains. Six compounds are more potent inhibitors than roscovitine, and three exhibited IC50 values close to 1 µM for both 3D7 and 7G8 strains. Although, such molecules do inhibit P. falciparum growth, they require further studies to improve their selectivity for PfCDKs.

  12. A single starfish Aurora kinase performs the combined functions of Aurora-A and Aurora-B in human cells.

    Science.gov (United States)

    Abe, Yusuke; Okumura, Eiichi; Hosoya, Takamitsu; Hirota, Toru; Kishimoto, Takeo

    2010-11-15

    Aurora, an essential mitotic kinase, is highly conserved during evolution. Most vertebrates have at least two Aurora kinases, Aurora-A and Aurora-B, which have distinct functions in the centrosome-spindle and inner centromere-midbody, respectively. However, some non-vertebrate deuterostomes have only a single Aurora. It remains to be verified whether the single Aurora performs the same functions as vertebrate Auroras A and B combined. We have isolated a cDNA of a single Aurora (ApAurora) from the echinoderm starfish, Asterina pectinifera, and show that ApAurora displays most features of both Aurora-A and Aurora-B in starfish oocytes and early embryos. Furthermore, ApAurora that is stably expressed in HeLa cells can substitute for both human Aurora-A and Aurora-B when either is reduced by RNAi. A single ApAurora thus has properties of both Aurora-A and Aurora-B in starfish eggs and HeLa cells. Together with phylogenetic analysis indicating that ApAurora forms a clade with all types of vertebrate Auroras and single Auroras of non-vertebrate deuterostomes, our observations support the idea that the single Aurora found in non-vertebrate deuterostomes represents the ancestor that gave rise to various types of vertebrate Auroras. This study thus provides functional evidence for phylogenetic considerations.

  13. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97.

    Science.gov (United States)

    Milbradt, Jens; Sonntag, Eric; Wagner, Sabrina; Strojan, Hanife; Wangen, Christina; Lenac Rovis, Tihana; Lisnic, Berislav; Jonjic, Stipan; Sticht, Heinrich; Britt, William J; Schlötzer-Schrehardt, Ursula; Marschall, Manfred

    2018-01-13

    The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  14. Src kinases are required for a balanced production of IL-12/IL-23 in human dendritic cells activated by Toll-like receptor agonists.

    Science.gov (United States)

    Kuka, Mirela; Baronio, Roberta; Valentini, Sara; Monaci, Elisabetta; Muzzi, Alessandro; Aprea, Susanna; De Gregorio, Ennio; D'Oro, Ugo

    2010-07-09

    Pathogen recognition by dendritic cells (DC) is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs) by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation. It was previously shown by our group that Src kinases play a key role in cytokines production during TLR4 activation in human DC. In this work we investigated the role of Src kinases during different TLRs triggering in human monocyte-derived DC (MoDC). We found that Src family kinases are important for a balanced production of inflammatory cytokines by human MoDC upon stimulation of TLR3 and 8 with their respective agonists. Disruption of this equilibrium through pharmacological inhibition of Src kinases alters the DC maturation pattern. In particular, while expression of IL-12 and other inflammatory cytokines depend on Src kinases, the induction of IL-23 and co-stimulatory molecules do not. Accordingly, DC treated with Src inhibitors are not compromised in their ability to induce CD4 T cell proliferation and to promote the Th17 subset survival but are less efficient in inducing Th1 differentiation. We suggest that the pharmacological modulation of DC maturation has the potential to shape the quality of the adaptive immune response and could be exploited for the treatment of inflammation-related diseases.

  15. The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells.

    Science.gov (United States)

    Martin del Campo, Sara E; Levine, Kala M; Mundy-Bosse, Bethany L; Grignol, Valerie P; Fairchild, Ene T; Campbell, Amanda R; Trikha, Prashant; Mace, Thomas A; Paul, Bonnie K; Jaime-Ramirez, Alena Cristina; Markowitz, Joseph; Kondadasula, Sri Vidya; Guenterberg, Kristan D; McClory, Susan; Karpa, Volodymyr I; Pan, Xueliang; Olencki, Thomas E; Monk, J Paul; Mortazavi, Amir; Tridandapani, Susheela; Lesinski, Gregory B; Byrd, John C; Caligiuri, Michael A; Shah, Manisha H; Carson, William E

    2015-09-01

    Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STAT1 and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR, and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 mg sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-α, and evaluated for phosphorylation of STAT1 and STAT5. Pretreatment of PBMCs with 10 μM sorafenib decreased STAT1 and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 μM), IL-2 (2-24 nM), and IFN-α (10(1)-10(6) U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MIP1-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells. Copyright © 2015 by The American Association of Immunologists, Inc.

  16. Regulation of glycogen synthase kinase-3 in human skeletal muscle: effects of food intake and bicycle exercise.

    Science.gov (United States)

    Wojtaszewski, J F; Nielsen, P; Kiens, B; Richter, E A; Wojtazsewski, J F

    2001-02-01

    Studies of skeletal muscle from rodents performed both in vivo and in vitro suggest a regulatory role of glycogen synthase kinase (GSK) 3 in glycogen synthase (GS) activation in response to insulin. Recently, hyperinsulinemic clamp studies in humans support such a role under nearly physiological conditions. In addition, in rats the activation of GS in skeletal muscle during treadmill running is time-related to the deactivation of GSK3. We investigated whether GSK3 was deactivated in human muscle during low- (approximately 50% VO2max for 1.5 h) and high-intensity (approximately 75% VO2max for 1 h) bicycle exercise as well as food intake. We observed a small but significant increase in GSK3alpha (10-20%) activity in biopsies obtained from vastus lateralis after both low- and high-intensity exercise, whereas GSK3beta activity was unaffected. Subsequent food intake increased Aktphosphorylation (approximately 2-fold) and deactivated GSK3alpha (approximately 40%), whereas GSK3beta activity was unchanged. GS activity increased in response to both exercise and food intake. We conclude that GSK3alpha but not GSK3beta may have a role in the regulation of GS activity in response to meal-associated hyperinsulinemia in humans. However, in contrast to findings in muscle from rats, exercise does not deactivate GSK3 in humans, suggesting a GSK3-independent mechanism in the regulation of GS activity in muscle during physical activity.

  17. An essential role of intestinal cell kinase in lung development is linked to the perinatal lethality of human ECO syndrome.

    Science.gov (United States)

    Tong, Yixin; Park, So Hyun; Wu, Di; Xu, Wenhao; Guillot, Stacey J; Jin, Li; Li, Xudong; Wang, Yalin; Lin, Chyuan-Sheng; Fu, Zheng

    2017-05-01

    Human endocrine-cerebro-osteodysplasia (ECO) syndrome, caused by the loss-of-function mutation R272Q in the intestinal cell kinase (ICK) gene, is a neonatal-lethal developmental disorder. To elucidate the molecular basis of ECO syndrome, we constructed an Ick R272Q knock-in mouse model that recapitulates ECO pathological phenotypes. Newborns bearing Ick R272Q homozygous mutations die at birth due to respiratory distress. Ick mutant lungs exhibit not only impaired branching morphogenesis associated with reduced mesenchymal proliferation but also significant airspace deficiency in primitive alveoli concomitant with abnormal interstitial mesenchymal differentiation. ICK dysfunction induces elongated primary cilia and perturbs ciliary Hedgehog signaling and autophagy during lung sacculation. Our study identifies an essential role for ICK in lung development and advances the mechanistic understanding of ECO syndrome. © 2017 Federation of European Biochemical Societies.

  18. 5'-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle

    DEFF Research Database (Denmark)

    Nielsen, Jakob Nis; Mustard, Kirsty J.W.; Graham, Drew A.

    2002-01-01

    (3)) AMPK subunits and exercise-induced AMPK activity are influenced by exercise training status, muscle biopsies were obtained from seven endurance exercise-trained and seven sedentary young healthy men. The alpha(1)- and alpha(2)-AMPK mRNA contents in trained subjects were both 117 +/- 2...... trained human skeletal muscle has increased alpha(1)-AMPK protein levels and blunted AMPK activation during exercise.......5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gamma...

  19. Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi.

    Science.gov (United States)

    Rispail, Nicolas; Soanes, Darren M; Ant, Cemile; Czajkowski, Robert; Grünler, Anke; Huguet, Romain; Perez-Nadales, Elena; Poli, Anna; Sartorel, Elodie; Valiante, Vito; Yang, Meng; Beffa, Roland; Brakhage, Axel A; Gow, Neil A R; Kahmann, Regine; Lebrun, Marc-Henri; Lenasi, Helena; Perez-Martin, José; Talbot, Nicholas J; Wendland, Jürgen; Di Pietro, Antonio

    2009-04-01

    Mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.

  20. The role of small adaptor proteins in the control of oncogenic signaling driven by tyrosine kinases in human cancer

    Science.gov (United States)

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-01-01

    Protein phosphorylation on tyrosine (Tyr) residues has evolved as an important mechanism to coordinate cell communication in multicellular organisms. The importance of this process has been revealed by the discovery of the prominent oncogenic properties of tyrosine kinases (TK) upon deregulation of their physiological activities, often due to protein overexpression and/or somatic mutation. Recent reports suggest that TK oncogenic signaling is also under the control of small adaptor proteins. These cytosolic proteins lack intrinsic catalytic activity and signal by linking two functional members of a catalytic pathway. While most adaptors display positive regulatory functions, a small group of this family exerts negative regulatory functions by targeting several components of the TK signaling cascade. Here, we review how these less studied adaptor proteins negatively control TK activities and how their loss of function induces abnormal TK signaling, promoting tumor formation. We also discuss the therapeutic consequences of this novel regulatory mechanism in human oncology. PMID:26788993

  1. The role of small adaptor proteins in the control of oncogenic signalingr driven by tyrosine kinases in human cancer.

    Science.gov (United States)

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-03-08

    Protein phosphorylation on tyrosine (Tyr) residues has evolved as an important mechanism to coordinate cell communication in multicellular organisms. The importance of this process has been revealed by the discovery of the prominent oncogenic properties of tyrosine kinases (TK) upon deregulation of their physiological activities, often due to protein overexpression and/or somatic mutation. Recent reports suggest that TK oncogenic signaling is also under the control of small adaptor proteins. These cytosolic proteins lack intrinsic catalytic activity and signal by linking two functional members of a catalytic pathway. While most adaptors display positive regulatory functions, a small group of this family exerts negative regulatory functions by targeting several components of the TK signaling cascade. Here, we review how these less studied adaptor proteins negatively control TK activities and how their loss of function induces abnormal TK signaling, promoting tumor formation. We also discuss the therapeutic consequences of this novel regulatory mechanism in human oncology.

  2. Erectile properties of the Rho-kinase inhibitor SAR407899 in diabetic animals and human isolated corpora cavernosa

    Science.gov (United States)

    2012-01-01

    Background RhoA-Rho kinase complex contributes to keep the cavernosus smooth muscle contracted and its inhibition is considered a potential strategy for the therapy of erectile dysfunction (ED). Methods We compared the effects of SAR407899, the Rho-kinase inhibitor Y-27632 and the PDE5 inhibitor sildenafil for their ability to relax corpus cavernosum strips contracted with phenylephrine in healthy and diabetic animals. Strips were obtained from WKY, spontaneous hypertensive (SHR), control CD, and diabetic CD rats, humans, control and diabetic rabbits. Diabetes was induced by streptozotocin or alloxan injection. In vivo penile erection (length) induced by drugs was measured in conscious rabbits. Results SAR407899 dose-dependently relaxed the pre-contracted corpora cavernosa in all species, with similar potency and efficacy in healthy vs diabetic rats, WKY vs SHR rats, healthy vs diabetic rabbits (IC50 range from 0.05 to 0.29 μM, Emax range 89 to 97%). In the presence of the NO-synthase (NOS) inhibitor, L-NAME, the SAR407899 response did not decrease in any of the species or experimental conditions. The effect was confirmed in human strips where sildenafil was significantly less potent and effective, with IC50 respectively 0.13 and 0.51 μM; Emax 92 and 43%. Unlike SAR407899, the potency and efficacy of sildenafil and Y27632 were significantly reduced by diabetes and L-NAME. In vivo, SAR407899 dose-dependently induced rabbit penile erection, with greater potency and longer duration of action than sildenafil. Sildenafil, but not SAR407899, was less effective in alloxan-induced diabetes. Conclusion The induction of penile erection by SAR407899, unlike that by sildenafil, is largely independent of e-NO activity. This suggests its use in erectile dysfunction for diabetic and hypertensive patients where e-NO activity is impaired. PMID:22444253

  3. Pfnek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum Biochemical properties and possible involvement in MAPK regulation.

    Science.gov (United States)

    Dorin, D; Le Roch, K; Sallicandro, P; Alano, P; Parzy, D; Poullet, P; Meijer, L; Doerig, C

    2001-05-01

    We have cloned Pfnek-1, a gene encoding a novel protein kinase from the human malaria parasite Plasmodium falciparum. This enzyme displays maximal homology to the never-in-mitosis/Aspergillus (NIMA)/NIMA-like kinase (Nek) family of protein kinases, whose members are involved in eukaryotic cell division processes. Similar to other P. falciparum protein kinases and many enzymes of the NIMA/Nek family, Pfnek-1 possesses a large C-terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfnek-1 protein is able to autophosphorylate and phosphorylate a panel of protein substrates with a specificity that is similar to that displayed by other members of the NIMA/Nek family. However, the FXXT motif usually found in NIMA/Nek protein kinases is substituted in Pfnek-1 by a SMAHS motif, which is reminiscent of a MAP/ERK kinase (MEK) activation site. Mutational analysis indicates that only one of the serine residues in this motif is essential for Pfnek-1 kinase activity in vitro. We show (a) that recombinant Pfnek-1 is able to specifically phosphorylate Pfmap-2, an atypical P. falciparum MAPK homologue, in vitro, and (b) that coincubation of Pfnek-1 and Pfmap-2 results in a synergistic increase in exogenous substrate labelling. This suggests that Pfnek-1 may be involved in the modulation of MAPK pathway output in malaria parasites. Finally, we demonstrate that recombinant Pfnek-1 can be used in inhibition assays to monitor the effect of kinase inhibitors, which opens the way to the screening of chemical libraries aimed at identifying potential new antimalarials.

  4. Janus-kinase-2 relates directly to portal hypertension and to complications in rodent and human cirrhosis.

    Science.gov (United States)

    Klein, Sabine; Rick, Johanna; Lehmann, Jennifer; Schierwagen, Robert; Schierwagen, Irela Gretchen; Verbeke, Len; Hittatiya, Kanishka; Uschner, Frank Erhard; Manekeller, Steffen; Strassburg, Christian P; Wagner, Kay-Uwe; Sayeski, Peter P; Wolf, Dominik; Laleman, Wim; Sauerbruch, Tilman; Trebicka, Jonel

    2017-01-01

    Angiotensin II (AngII) activates via angiotensin-II-type-I receptor (AT1R) Janus-kinase-2 (JAK2)/Arhgef1 pathway and subsequently RHOA/Rho-kinase (ROCK), which induces experimental and probably human liver fibrosis. This study investigated the relationship of JAK2 to experimental and human portal hypertension. The mRNA and protein levels of JAK2/ARHGEF1 signalling components were analysed in 49 human liver samples and correlated with clinical parameters of portal hypertension in these patients. Correspondingly, liver fibrosis (bile duct ligation (BDL), carbon tetrachloride (CCl4)) was induced in floxed-Jak2 knock-out mice with SM22-promotor (SM22(Cre+)-Jak2(f/f)). Transcription and contraction of primary myofibroblasts from healthy and fibrotic mice and rats were analysed. In two different cirrhosis models (BDL, CCl4) in rats, the acute haemodynamic effect of the JAK2 inhibitor AG490 was assessed using microsphere technique and isolated liver perfusion experiments. Hepatic transcription of JAK2/ARHGEF1 pathway components was upregulated in liver cirrhosis dependent on aetiology, severity and complications of human liver cirrhosis (Model for End-stage Liver disease (MELD) score, Child score as well as ascites, high-risk varices, spontaneous bacterial peritonitis). SM22(Cre+)- Jak2(f/f) mice lacking Jak2 developed less fibrosis and lower portal pressure (PP) than SM22(Cre-)-Jak2(f/f) upon fibrosis induction. Myofibroblasts from SM22(Cre+)-Jak2(f/f) mice expressed less collagen and profibrotic markers upon activation. AG490 relaxed activated hepatic stellate cells in vitro. In cirrhotic rats, AG490 decreased hepatic vascular resistance and consequently the PP in vivo and in situ. Hepatic JAK2/ARHGEF1/ROCK expression is associated with portal hypertension and decompensation in human cirrhosis. The deletion of Jak2 in myofibroblasts attenuated experimental fibrosis and acute inhibition of JAK2 decreased PP. Thus, JAK2 inhibitors, already in clinical use for other

  5. TMPYP4 exerted antitumor effects in human cervical cancer cells through activation of p38 mitogen-activated protein kinase.

    Science.gov (United States)

    Cheng, Ming-Jun; Cao, Yun-Gui

    2017-07-03

    The aim of the present study was to investigate the potential effects of the 5,10,15,20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) on the proliferation and apoptosis of human cervical cancer cells and the underlying mechanisms by which TMPyP4 exerted its actions. After human cervical cancer cells were treated with different doses of TMPyP4, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method, the apoptosis was observed by flow cytometry (FCM), and the expression of p38 mitogen-activated protein kinase (MAPK), phosphated p38 MAPK (p-p38 MAPK), capase-3, MAPKAPK2 (MK-2) and poly ADP-ribose polymerase (PARP) was measured by Western blot analysis. The analysis revealed that TMPyP4 potently suppressed cell viability and induced the apoptosis of human cervical cancer cells in a dose-dependent manner. In addition, the up-regulation of p-p38 MAPK expression levels was detected in TMPyP4-treated human cervical cancer cells. However, followed by the block of p38 MAPK signaling pathway using the inhibitor SB203580, the effects of TMPyP4 on proliferation and apoptosis of human cervical cancer cells were significantly changed. It was indicated that TMPyP4-inhibited proliferation and -induced apoptosis in human cervical cancer cells was accompanied by activating the p38 MAPK signaling pathway. Taken together, our study demonstrates that TMPyP4 may represent a potential therapeutic method for the treatment of cervical carcinoma.

  6. Growth factor-induced contraction of human bronchial smooth muscle is Rho-kinase-dependent

    NARCIS (Netherlands)

    Gosens, Reinout; Schaafsma, D.; Grootte Bromhaar, M.M; Vrugt, B.; Zaagsma, Hans; Meurs, Herman; Nelemans, Herman

    2004-01-01

    Growth factors have been implicated in the pathophysiology of asthma. However, the putative effects of these growth factors on human airway smooth muscle tone are still largely unknown. We performed contraction experiments using human bronchial smooth muscle ring preparations. The growth factor

  7. A Murine Model for Human ECO Syndrome Reveals a Critical Role of Intestinal Cell Kinase in Skeletal Development.

    Science.gov (United States)

    Ding, Mengmeng; Jin, Li; Xie, Lin; Park, So Hyun; Tong, Yixin; Wu, Di; Chhabra, A Bobby; Fu, Zheng; Li, Xudong

    2017-11-02

    An autosomal-recessive inactivating mutation R272Q in the human intestinal cell kinase (ICK) gene caused profound multiplex developmental defects in human endocrine-cerebro-osteodysplasia (ECO) syndrome. ECO patients exhibited a wide variety of skeletal abnormalities, yet the underlying mechanisms by which ICK regulates skeletal development remained largely unknown. The goal of this study was to understand the structural and mechanistic basis underlying skeletal anomalies caused by ICK dysfunction. Ick R272Q knock-in transgenic mouse model not only recapitulated major ECO skeletal defects such as short limbs and polydactyly but also revealed a deformed spine with defective intervertebral disk. Loss of ICK function markedly reduced mineralization in the spinal column, ribs, and long bones. Ick mutants showed a significant decrease in the proliferation zone of long bones and the number of type X collagen-expressing hypertrophic chondrocytes in the spinal column and the growth plate of long bones. These results implicate that ICK plays an important role in bone and cartilage development by promoting chondrocyte proliferation and maturation. Our findings provided new mechanistic insights into the skeletal phenotype of human ECO and ECO-like syndromes.

  8. Structural Properties of Human CaMKII Ca2+ /Calmodulin-Dependent Protein Kinase II using X-ray Crystallography

    Science.gov (United States)

    Cao, Yumeng Melody; McSpadden, Ethan; Kuriyan, John; Department of Molecular; Cell Biology; Department of Chemistry Team

    To this day, human memory storage remains a mystery as we can at most describe the process vaguely on a cellular level. Switch-like properties of Calcium/Calmodulin-Dependent Protein Kinase II make it a leading candidate in understanding the molecular basis of human memory. The protein crystal was placed in the beam of a synchrotron source and the x-ray crystallography data was collected as reflections on a diffraction pattern that undergo Fourier transform to obtain the electron density. We observed two drastic differences from our solved structure at 2.75Å to a similar construct of the mouse CaMKII association domain. Firstly, our structure is a 6-fold symmetric dodecamer, whereas the previously published construct was a 7-fold symmetric tetradecamer. This suggests the association domain of human CaMKII is a dynamic structure that is triggered subunit exchange process. Secondly, in our structure the N-terminal tag is docked as an additional beta-strand on an uncapped beta-sheet present in each association domain protomer. This is concrete evidence of the involvement of the polypeptide docking site in the molecular mechanism underlining subunit exchange. In the future, we would like to selectively inhibit the exchange process while not disrupting the other functionalities of CaMKII.

  9. Regulation of Discrete Functional Responses by Syk and Src Family Tyrosine Kinases in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Thornin Ear

    2017-01-01

    Full Text Available Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs, as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates (e.g., eIF-4E, S6K, and MNK1 involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.

  10. Rho kinase inhibitor y-27632 prolongs the life span of adult human keratinocytes, enhances skin equivalent development, and facilitates lentiviral transduction.

    NARCIS (Netherlands)

    Bogaard, E.H.J. van den; Rodijk-Olthuis, D.; Jansen, P.A.M.; Vlijmen-Willems, I.M.J.J. van; Erp, P.E.J. van; Joosten, I.; Zeeuwen, P.L.J.M.; Schalkwijk, J.

    2012-01-01

    The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases,

  11. Spatial distribution of "tissue-specific" antigens in the developing human heart and skeletal muscle. I. An immunohistochemical analysis of creatine kinase isoenzyme expression patterns

    NARCIS (Netherlands)

    Wessels, A.; Vermeulen, J. L.; Virágh, S.; Kálmán, F.; Morris, G. E.; Man, N. T.; Lamers, W. H.; Moorman, A. F.

    1990-01-01

    Using monoclonal antibodies against the M and B subunit isoforms of creatine kinase (CK) we have investigated their distribution in developing human skeletal and cardiac muscle immunohistochemically. It is demonstrated that in skeletal muscle, a switch from CK-B to CK-M takes place around the week 8

  12. Pasteurella multocida thymidine kinase 1 efficiently activates pyrimidine nucleoside analogs.

    Science.gov (United States)

    Clausen, A R; Al Meani, S A L; Piskur, J

    2010-06-01

    In the Pasteurella multocida genome only one putative deoxyribonucleoside kinase encoding gene, for thymidine kinase 1 (PmTK1), was identified. The PmTK1 gene was sub-cloned into Escherichia coli KY895 and it sensitized the host towards 2',2'-difluoro-deoxycytidine (gemcitabine, dFdC), 3'-azido-thymidine (AZT) and 5-fluoro-deoxyuridine (5F-dU). PmTK1 was over-expressed and purified with two different tags. Apparently, deoxyuridine (dU), and not thymidine (dT), is the preferred substrate. We suggest that PmTK1s could be employed as a species-specific activator of uracil-based nucleoside antibiotics.

  13. Distribution and Evolution of CTG Repeats at the Myotonin Protein Kinase Gene in Human Populations

    OpenAIRE

    Deka, Ranjan; Majumder, Partha P.; Shriver, Mark D.; Stivers, David N.; Zhong, Yixi; Yu, Ling M.; Barrantes-Mesén, Ramiro; Yin, Shih-Jiun; Miki, Tetsuro; Hundrieser, Joachim; Bunker, Clareann H.; McGarvey, Stephen T.; Sakallah, Sameer; Ferrell, Robert E.; Chakraborty, Ranajit

    1996-01-01

    Artículo científico -- Instituto de Investigaciones en Salud. 1996 We have analyzed the CTG repeat length and the neighboring Alu insertion/deletion (+/-) polymorphism in DNA samples from 16 ethnically and geographically diverse human populations to understand the evolutionary dynamics of the myotonic dystrophy-associated CTG repeat. Our results show that the CTG repeat length is variable in human populations. Although the (CTG)5 repeat is the most common allele in the majority of populati...

  14. DNA-hypomethylating agent, 5'-azacytidine, induces cyclooxygenase-2 expression via the PI3-kinase/Akt and extracellular signal-regulated kinase-1/2 pathways in human HT1080 fibrosarcoma cells.

    Science.gov (United States)

    Yu, Seon-Mi; Kim, Song-Ja

    2015-10-01

    The cytosine analogue 5'-azacytidine (5'-aza) induces DNA hypomethylation by inhibiting DNA methyltransferase. In clinical trials, 5'-aza is widely used in epigenetic anticancer treatments. Accumulated evidence shows that cyclooxygenase-2 (COX-2) is overexpressed in various cancers, indicating that it may play a critical role in carcinogenesis. However, few studies have been performed to explore the molecular mechanism underlying the increased COX-2 expression. Therefore, we tested the hypothesis that 5'-aza regulates COX-2 expression and prostaglandin E2 (PGE2) production. The human fibrosarcoma cell line HT1080, was treated with various concentrations of 5'-aza for different time periods. Protein expressions of COX-2, DNA (cytosine-5)-methyltransferase 1 (DNMT1), pAkt, Akt, extracellular signal-regulated kinase (ERK), and phosphorylated ERK (pERK) were determined using western blot analysis, and COX-2 mRNA expression was determined using RT-PCR. PGE2 production was evaluated using the PGE2 assay kit. The localization and expression of COX-2 were determined using immunofluorescence staining. Treatment with 5'-aza induces protein and mRNA expression of COX-2. We also observed that 5'-aza-induced COX-2 expression and PGE2 production were inhibited by S-adenosylmethionine (SAM), a methyl donor. Treatment with 5'-aza phosphorylates PI3-kinase/Akt and ERK-1/2; inhibition of these pathways by LY294002, an inhibitor of PI3-kinase/Akt, or PD98059, an inhibitor of ERK-1/2, respectively, prevents 5'-aza-induced COX-2 expression and PGE2 production. Overall, these observations indicate that the hypomethylating agent 5'-aza modulates COX-2 expression via the PI3-kinase/Akt and ERK-1/2 pathways in human HT1080 fibrosarcoma cells.

  15. The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models of Human Neuroblastoma.

    Science.gov (United States)

    Lowery, Caitlin D; VanWye, Alle B; Dowless, Michele; Blosser, Wayne; Falcon, Beverly L; Stewart, Julie; Stephens, Jennifer; Beckmann, Richard P; Bence Lin, Aimee; Stancato, Louis F

    2017-08-01

    Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G2-M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma.Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line-derived xenograft mouse models of neuroblastoma.Results: Within 24 hours, single-agent prexasertib promoted γH2AX-positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA-PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature.Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354-63. ©2017 AACR. ©2017 American Association for Cancer Research.

  16. Prognostic Value of Focal Adhesion Kinase (FAK in Human Solid Carcinomas: A Meta-Analysis.

    Directory of Open Access Journals (Sweden)

    Xiao-Qing Zeng

    Full Text Available Recently, the number of reports on focal adhesion kinase (FAK as a vital therapeutic target in solid carcinomas has increased; however, the prognostic role of FAK status remains poorly understood. This study aims to evaluate the prognostic effect of FAK by means of a meta-analysis.We performed a systematic literature search in order to examine the correlation between expression of FAK and overall survival(OS. The hazard ratio (HR of OS was used to measure survival. A random-effects model was used to pool study statistics. Sensitivity and publication bias analyses were also conducted.Thirty eligible studies involving 4702 patients were included. The median expression rate of FAK was 54%. Meta-analysis of the HRs demonstrated that high FAK expression was associated with worse OS (average HR = 2.073, 95%confidence interval[CI]:1.712-2.510, p = 0.000. Regarding cancer type, FAK was associated with worse OS in gastric cancer (HR = 2.646,95% CI:1.743-4.017, p = 0.000, hepatocellular carcinoma (HR = 1.788,95% CI:1.228-2.602, p = 0.002, ovarian cancer (HR = 1.815, 95% CI: 1.193-2.762, p = 0.005, endometrial cancer (HR = 4.149, 95% CI:2.832-6.079, p = 0.000, gliomas (HR = 2.650, 95% CI: 1.205-5.829, p = 0.015, and squamous cell carcinoma (HR = 1,696, 95% CI: 1.030-2.793, p = 0.038. No association was found between HR and disease staging according to our meta-regression analysis.Our study shows that high expression of FAK is associated with a worse OS in patients with carcinomas, but the association between FAK and prognosis varies according to cancer type. The value of FAK status in clinical prognosis in cancer needs further research.

  17. Effect of Biodentine and Bioaggregate on odontoblastic differentiation via mitogen-activated protein kinase pathway in human dental pulp cells.

    Science.gov (United States)

    Jung, J-Y; Woo, S-M; Lee, B-N; Koh, J-T; Nör, J E; Hwang, Y-C

    2015-02-01

    To compare the mineralization inductive capacity of Biodentine and Bioaggregate with Mineral trioxide aggregate (MTA) and to investigate possible signaling pathways of mineralization in human dental pulp cells (HDPCs). Viability of HDPCs in response to Biodentine, Bioaggregate, and MTA was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide. To investigate their potential to induce odontoblast differentiation, expression of dentine sialophosphoprotein (DSPP) and dentine matrix protein1 (DMP1) mRNA level was evaluated by RT-PCR. For the mineralized nodule assay, Alizarin red staining was performed. To determine the role of MAPK signaling in the odontoblastic differentiation of HDPCs, activated MAPKs were investigated by Western blot and the effect of MAPK inhibitor was examined by Alizarin red S staining. The results were statistically analysed using one-way anova and the Bonferroni test. The effects of MTA, Biodentine, and Bioaggregate on cell viability were similar. Biodentine and Bioaggregate enhanced DSPP and DMP1 mRNA expression compared to the control group, but to the same extent as MTA (P Biodentine, and Bioaggregate increased the area of calcified nodules compared to the control (P Biodentine, and Bioaggregate increased phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). MAPK inhibitors attenuated mineralized nodule formation, which was increased by MTA, Biodentine, and Bioaggregate, respectively (P Biodentine and Bioaggregate stimulated odontoblastic differentiation and mineralization nodule formation by activating the MAPK pathway as did MTA. This suggests that the new materials could be useful for regenerative endodontic procedures. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  18. Curcumin specifically binds to the human calcium-calmodulin-dependent protein kinase IV: fluorescence and molecular dynamics simulation studies.

    Science.gov (United States)

    Hoda, Nasimul; Naz, Huma; Jameel, Ehtesham; Shandilya, Ashutosh; Dey, Sharmistha; Hassan, Md Imtaiyaz; Ahmad, Faizan; Jayaram, B

    2016-01-01

    Calcium-calmodulin-dependent protein kinase IV (CAMK4) plays significant role in the regulation of calcium-dependent gene expression, and thus, it is involved in varieties of cellular functions such as cell signaling and neuronal survival. On the other hand, curcumin, a naturally occurring yellow bioactive component of turmeric possesses wide spectrum of biological actions, and it is widely used to treat atherosclerosis, diabetes, cancer, and inflammation. It also acts as an antioxidant. Here, we studied the interaction of curcumin with human CAMK4 at pH 7.4 using molecular docking, molecular dynamics (MD) simulations, fluorescence binding, and surface plasmon resonance (SPR) methods. We performed MD simulations for both neutral and anionic forms of CAMK4-curcumin complexes for a reasonably long time (150 ns) to see the overall stability of the protein-ligand complex. Molecular docking studies revealed that the curcumin binds in the large hydrophobic cavity of kinase domain of CAMK4 through several hydrophobic and hydrogen-bonded interactions. Additionally, MD simulations studies contributed in understanding the stability of protein-ligand complex system in aqueous solution and conformational changes in the CAMK4 upon binding of curcumin. A significant increase in the fluorescence intensity at 495 nm was observed (λexc = 425 nm), suggesting a strong interaction of curcumin to the CAMK4. A high binding affinity (KD = 3.7 × 10(-8) ± .03 M) of curcumin for the CAMK4 was measured by SPR further indicating curcumin as a potential ligand for the CAMK4. This study will provide insights into designing a new inspired curcumin derivatives as therapeutic agents against many life-threatening diseases.

  19. Conserved retinoblastoma protein-binding motif in human cytomegalovirus UL97 kinase minimally impacts viral replication but affects susceptibility to maribavir

    Directory of Open Access Journals (Sweden)

    Chou Sunwen

    2009-01-01

    Full Text Available Abstract The UL97 kinase has been shown to phosphorylate and inactivate the retinoblastoma protein (Rb and has three consensus Rb-binding motifs that might contribute to this activity. Recombinant viruses containing mutations in the Rb-binding motifs generally replicated well in human foreskin fibroblasts with only a slight delay in replication kinetics. Their susceptibility to the specific UL97 kinase inhibitor, maribavir, was also examined. Mutation of the amino terminal motif, which is involved in the inactivation of Rb, also renders the virus hypersensitive to the drug and suggests that the motif may play a role in its mechanism of action.

  20. Functional characterization of human RSK4, a new 90-kDa ribosomal S6 kinase, reveals constitutive activation in most cell types

    DEFF Research Database (Denmark)

    Dümmler, Bettina A; Hauge, Camilla; Silber, Joachim

    2005-01-01

    characterization of a predicted new human RSK homologue, RSK4. We showed that RSK4 is a predominantly cytosolic protein with very low expression and several characteristics of the RSK family kinases, including the presence of two functional kinase domains and a C-terminal docking site for ERK. Surprisingly......, however, in all cell types analyzed, endogenous RSK4 was maximally (constitutively) activated under serum-starved conditions where other RSKs are inactive due to their requirement for growth factor stimulation. Constitutive activation appeared to result from constitutive phosphorylation of Ser232, Ser372...

  1. Theoretical studies of the role of C-terminal cysteines in the process of S-nitrosylation of human Src kinases.

    Science.gov (United States)

    Andre, Fernanda R; dos Santos, Paloma Freire; Rando, Daniela G

    2016-01-01

    Src tyrosine kinases are a family of non-receptor proteins that are responsible for the growth process, cellular proliferation, differentiation and survival. Lack of Src kinase control has been associated with the development of certain human cancers. This family of proteins is constituted of four domains, with SH1 being the kinase or catalytic domain. SH1 also presents three important regulatory sites. Two residues, Tyr416 and Tyr527, are responsible for important phosphorylation processes that lead to, respectively, activation and deactivation of these kinases. More recently, however, a set of four cysteine residues located near the C-terminus-Cys483, Cys487, Cys496 and Cys498-has been associated with the activation of the Src kinases through S-nitrosylation reactions. Particularly, the Cys498 has been specified as a fundamental residue when considering this regulatory mechanism. Aiming to understand the role of these four cysteines in S-nitrosylation, theoretical studies of electrostatic, steric and hydrophobic properties were performed with a sequence of 20 amino acids, enclosing the four cysteine residues under study, extracted from the PDB coordinates of the crystal obtained from the inactive state of Src kinase. Results indicate that Cys498 is buried deeply in the protein, in hydrophobic surroundings in which NO is more likely to suffer decomposition into the electrophilic intermediates known to be responsible for S-nitrosylation reactions. Electronic calculated properties, such as punctual atomic charges, electrostatic potentials and molecular orbital energy, also demonstrated the good nucleophilic potential of Cys498. Graphical Abstract Structure of Src kinase with zoomed area representing the 20 amino acids comprising the CC motif extracted from the whole protein structure. Right upper panel Electrostatic potential map, right lower panel hydrophilic map in anterior view.

  2. Protein kinase D regulates the human cardiac L-type voltage-gated calcium channel through serine 1884.

    Science.gov (United States)

    Aita, Yusuke; Kurebayashi, Nagomi; Hirose, Shigehisa; Maturana, Andrés D

    2011-12-15

    Protein kinase D (PKD) regulates the activity of the L-type calcium channel in rat ventricular cardiomyocytes. However, the functional target residues of PKD on the L-type calcium channel remain to be identified. Our aim was to identify the functional phosphorylation sites of PKD on the human L-type calcium channel. The pore subunit of the human CaV1.2 (hCaV1.2) was stably expressed in HEK293 cells. Both the expression of a dominant-negative mutant of PKD and the mutation of serine 1884 but not serine 1930, putative targets of PKD, strongly reduced L-type calcium currents and single channel activity without affecting the channel's expression at the plasma membrane. Our results suggest that serine 1884 is essential for the regulation of hCaV1.2 by PKD. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Inhibition of p21 activated kinase (PAK reduces airway responsiveness in vivo and in vitro in murine and human airways.

    Directory of Open Access Journals (Sweden)

    Wyn C Hoover

    Full Text Available The p21-activated protein kinases (Paks have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak1(-/- was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh. Pulmonary resistance was measured in anesthetized mechanically ventilated Pak1(-/- and wild type mice. Pak1(-/- mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak1(-/- mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness.

  4. Bioelectrocatalytic sensor for triglycerides in human skin sebum based on enzymatic cascade reaction of lipase, glycerol kinase and glycerophosphate oxidase.

    Science.gov (United States)

    Jeong, Chi Yong; Han, Yong Duk; Yoon, Jae Ho; Yoon, Hyun C

    2014-04-10

    We report the development of an electrochemical biosensor for the quantification of triglycerides in human skin sebum, based on a multienzyme cascade reaction. The presence of excessive triglycerides in human sebum is one of the leading causes of various skin ailments. However, to the best of our knowledge, no bioelectrocatalytic approach for the quantification of sebum triglycerides has been made. In order to develop triglyceride biosensor, we fabricated a multienzyme-associated electrode incorporating lipase, glycerol kinase, and glycerophosphate oxidase. Enzymes were deposited by electrostatic force and further stabilized via crosslinking between enzymes and polymer matrices. The enzyme-modified biosensing electrode maintained its bioelectrocatalytic activity for five days. An additional constraint was the limited solubility of sebum triglycerides in aqueous electrolytes, impeding the analysis. To address this issue, triglyceride samples were prepared in the form of micelles, enabling efficient sample preparation for biosensor signaling. Calibration tests revealed that the designed assay had a detection range of 15-200mg/dL of micellar triglyceride, which covered the required determination range. The developed biosensing approach was successfully used to determine triglyceride concentrations in real sebum samples of unknown triglyceride content. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Human cytomegalovirus UL97 kinase is involved in the mechanism of action of methylenecyclopropane analogs with 6-ether and -thioether substitutions.

    Science.gov (United States)

    Komazin-Meredith, Gloria; Chou, Sunwen; Prichard, Mark N; Hartline, Caroll B; Cardinale, Steven C; Comeau, Katelyn; Williams, John D; Khan, Atiyya R; Peet, Norton P; Bowlin, Terry L

    2014-01-01

    Methylenecyclopropane nucleoside (MCPN) analogs are being investigated for treatment of human cytomegalovirus (HCMV) infection because of favorable preclinical data and limited ganciclovir cross-resistance. Monohydroxymethyl MCPNs bearing ether and thioether functionalities at the purine 6 position have antiviral activity against herpes simplex virus (HSV) and varicella-zoster virus (VZV) in addition to HCMV. The role of the HCMV UL97 kinase in the mechanism of action of these derivatives was examined. When tested against a kinase-inactive UL97 K355M virus, a moderate 5- to 7-fold increase in 50% effective concentration (EC50) was observed, in comparison to a 13- to 25-fold increase for either cyclopropavir or ganciclovir. Serial propagation of HCMV under two of these compounds selected for three novel UL97 mutations encoding amino acid substitutions D456N, C480R,and Y617del. When transferred to baseline laboratory HCMV strains, these mutations individually conferred resistance to all of the tested MCPNs, ganciclovir, and maribavir. However, the engineered strains also demonstrated severe growth defects and abnormal cytopathic effects similar to the kinase-inactive mutant. Expressed and purified UL97 kinase showed in vitro phosphorylation of the newly tested MCPNs. Thus, HCMV UL97 kinase is involved in the antiviral action of these MCPNs, but the in vitro selection of UL97-defective viruses suggests that their activity against more typical ganciclovir-resistant growth-competent UL97 mutants may be relatively preserved.

  6. Cloning and partial characterization of the human tie-2 receptor tyrosine kinase gene promoter.

    Science.gov (United States)

    Hewett, P W; Daft, E L; Murray, J C

    1998-11-27

    The Tie-2 receptor plays a key role in vascular development, although little is known about the factors controlling its expression. Here we report the first cloning and characterisation of the 5' regulatory region of human tie-2. Multiple transcription start sites were identified between -414 and -265 bp upstream of the start codon using 5' RACE, fluorescent primer extension, and RNase protection assays. The human tie-2 promoter contains several transcription factor-binding sequences including ets, SP-1, AP-1, and GATA-1, but there are no canonical TATA or CCAAT initiation sequences proximal to the transcription start sites. Human tie-2 reporter constructs demonstrated approximately 10-fold greater activity in endothelial cells compared with fibroblasts. In endothelial cells the tie-2 promoter exhibited 5 and 16% of the activity of human tie-1 (830 bp) and KDR (1.1 kb) promoters, respectively. This promoter will be a useful tool for studying factors that regulate tie-2 expression and targeting the vasculature. Copyright 1998 Academic Press.

  7. A point mutation at tyrosine-809 in the human colony-stimulating factor 1 receptor impairs mitogenesis without abrogating tyrosine kinase activity, association with phosphatidylinositol 3-kinase, or induction of c-fos and junB genes

    Energy Technology Data Exchange (ETDEWEB)

    Roussel, M.F. (Univ. of Tennessee, Memphis (USA)); Shurtleff, S.A.; Downing, J.R. (Saint Jude Children' s Research Hospital, Memphis, TN (USA)); Sherr, C.J. (Univ. of Tennessee College of Medicine, Memphis (USA) Saint Jude Children' s Research Hospital, Memphis, TN (USA))

    1990-09-01

    Substitution of phenylalanine for tyrosine-809 in the human colony-stimulating factor 1 receptor (CSF-1R) inhibited its ability to transduce ligand-dependent mitogenic signals in mouse NIH 3T3 cells. When combined with an activating mutation at codon 301 that induces constitutive CSF-1R tyrosine kinase activity, the codon 809 mutation suppressed ligand-independent cell transformation. Comparative mapping tryptic phosphopeptides from mutant and wild-type CSF-1R indicated that tyrosine-809 is a site of ligand-dependent receptor phosphorylation in vivo. The mutant receptor was active as a tyrosine kinase in vitro and in vivo, underwent CSF-1-dependent association with a phosphatidylinositol 3-kinase, and induced expression of the protooncogenes c-fos and junB, underscoring its ability to trigger some of the known cellular responses to CSF-1. The mutant receptor is likely to be impaired in its ability to interact with critical cellular effectors whose activity is required for mitogenesis.

  8. Chemical genomic screening for methylation-silenced genes in gastric cancer cell lines using 5-aza-2-deoxycytidine treatment and oligonucleotide microarray

    National Research Council Canada - National Science Library

    Yamashita, Satoshi; Tsujino, Yoshimi; Moriguchi, Kazuki; Tatematsu, Masae; Ushijima, Toshikazu

    2006-01-01

    To identify novel methylation-silenced genes in gastric cancers, we carried out a chemical genomic screening, a genome-wide search for genes upregulated by treatment with a demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC...

  9. AN ESCHERICHIA-COLI STRAIN DEFICIENT FOR BOTH EXONUCLEASE-V AND DEOXYCYTIDINE TRIPHOSPHATE DEAMINASE SHOWS ENHANCED SENSITIVITY TO IONIZING-RADIATION

    NARCIS (Netherlands)

    ESTEVENON, AM; KOOISTRA, J; SICARD, N

    1995-01-01

    An Escherichia coli mutant lacking deoxycytidine triphosphate deaminase (Dcd) activity and an unknown function encoded by a gene designated ior exhibits sensitivity to ionizing radiation whereas dcd mutants themselves are not sensitive. A DNA fragment from an E. coli genomic library that restores

  10. Effects of Acrylamide on the Activity and Structure of Human Brain Creatine Kinase

    Science.gov (United States)

    Sheng, Qing; Zou, He-Chang; Lü, Zhi-Rong; Zou, Fei; Park, Yong-Doo; Yan, Yong-Bin; Yao, Shan-Jing

    2009-01-01

    Acrylamide is widely used worldwide in industry and it can also be produced by the cooking and processing of foods. It is harmful to human beings, and human brain CK (HBCK) has been proposed to be one of the important targets of acrylamide. In this research, we studied the effects of acrylamide on HBCK activity, structure and the potential binding sites. Compared to CKs from rabbit, HBCK was fully inactivated at several-fold lower concentrations of acrylamide, and exhibited distinct properties upon acrylamide-induced inactivation and structural changes. The binding sites of acrylamide were located at the cleft between the N- and C-terminal domains of CK, and Glu232 was one of the key binding residues. The effects of acrylamide on CK were proposed to be isoenzyme- and species-specific, and the underlying molecular mechanisms were discussed. PMID:20057941

  11. Genes influenced by the non-muscle isoform of Myosin light chain kinase impact human cancer prognosis.

    Directory of Open Access Journals (Sweden)

    Tong Zhou

    Full Text Available The multifunctional non-muscle isoform of myosin light chain kinase (nmMLCK is critical to the rapid dynamic coordination of the cytoskeleton involved in cancer cell proliferation and migration. We identified 45 nmMLCK-influenced genes by bioinformatic filtering of genome-wide expression in wild type and nmMLCK knockout (KO mice exposed to preclinical models of murine acute inflammatory lung injury, pathologies that are well established to include nmMLCK as an essential participant. To determine whether these nmMLCK-influenced genes were relevant to human cancers, the 45 mouse genes were matched to 38 distinct human orthologs (M38 signature (GeneCards definition and underwent Kaplan-Meier survival analysis in training and validation cohorts. These studies revealed that in training cohorts, the M38 signature successfully identified cancer patients with poor overall survival in breast cancer (P<0.001, colon cancer (P<0.001, glioma (P<0.001, and lung cancer (P<0.001. In validation cohorts, the M38 signature demonstrated significantly reduced overall survival for high-score patients of breast cancer (P = 0.002, colon cancer (P = 0.035, glioma (P = 0.023, and lung cancer (P = 0.023. The association between M38 risk score and overall survival was confirmed by univariate Cox proportional hazard analysis of overall survival in the both training and validation cohorts. This study, providing a novel prognostic cancer gene signature derived from a murine model of nmMLCK-associated lung inflammation, strongly supports nmMLCK-involved pathways in tumor growth and progression in human cancers and nmMLCK as an attractive candidate molecular target in both inflammatory and neoplastic processes.

  12. Antitumor activity of sorafenib in human cancer cell lines with acquired resistance to EGFR and VEGFR tyrosine kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Floriana Morgillo

    Full Text Available Treatment of non small cell lung cancer (NSCLC and colorectal cancer (CRC have substantially changed in the last years with the introduction of epidermal growth factor receptor (EGFR inhibitors in the clinical practice. The understanding of mechanisms which regulate cells sensitivity to these drugs is necessary for their optimal use.An in vitro model of acquired resistance to two tyrosine kinase inhibitors (TKI targeting the EGFR, erlotinib and gefitinib, and to a TKI targeting EGFR and VEGFR, vandetanib, was developed by continuously treating the human NSCLC cell line CALU-3 and the human CRC cell line HCT116 with escalating doses of each drug. MTT, western blot analysis, migration, invasion and anchorage-independent colony forming assays were conducted in vitro and experiments with established xenografts in athymic nude mice were performed in vivo in sensitive, wild type (WT and TKI-resistant CALU-3 and HCT116 cell lines.As compared to WT CALU-3 and HCT116 human cancer cells, TKI-resistant cell lines showed a significant increase in the levels of activated, phosphorylated AKT, MAPK, and of survivin. Considering the role of RAS and RAF as downstream signals of both the EGFR and VEGFR pathways, we treated resistant cells with sorafenib, an inhibitor of C-RAF, B-RAF, c-KIT, FLT-3, RET, VEGFR-2, VEGFR-3, and PDGFR-β. Sorafenib reduced the activation of MEK and MAPK and caused an inhibition of cell proliferation, invasion, migration, anchorage-independent growth in vitro and of tumor growth in vivo of all TKI-resistant CALU-3 and HCT116 cell lines.These data suggest that resistance to EGFR inhibitors is predominantly driven by the RAS/RAF/MAPK pathway and can be overcame by treatment with sorafenib.

  13. In Vitro Inhibition of Human Sperm Creatine Kinase by Nicotine,Cotinine and Cadmium, as a Mechanism in Smoker Men Infertility

    Directory of Open Access Journals (Sweden)

    Mohammad Ali Ghaffari

    2009-01-01

    Full Text Available Background: Nicotine, cotinine and cadmium are harmful components of cigarettes that have aneffect on human reproductive function. Although the effects of cigarette smoke on male reproductivefunction is characterized in several articles its mechanism of action is still unknown.In the present study, we investigate the effect of nicotine, cotinine and cadmium on human spermcreatine kinase activity in vitro.Materials and Methods: Total creatine kinase activity is measured in sperm homogenates afterchromatography on a diethylaminoethyl cellulose (DEAE-32 column.Results: We show that creatine kinase activity is significantly inhibited by nicotine (44%, cotinine(39% and cadmium (65% at a concentration of 60 μg/ml. Kinetic studies reveal that the inhibitoryeffect of nicotine, cotinine and cadmium are competitive in relation to creatine phosphate.Conclusion: Considering the importance of creatine kinase activity for normal sperm energymetabolism, our results suggest that inhibition of this enzyme by nicotine, cotinine and cadmium maybe an important mechanism in infertility amongst male smokers. However, further investigationsare needed to elucidate the exact mechanism of cigarette effect on male reproductive function atthe molecular level.

  14. Expression of serine/threonine protein-kinases and related factors in normal monkey and human retinas: the mechanistic understanding of a CDK2 inhibitor induced retinal toxicity.

    Science.gov (United States)

    Saturno, Grazia; Pesenti, Manuela; Cavazzoli, Cristiano; Rossi, Anna; Giusti, Anna M; Gierke, Berthold; Pawlak, Michael; Venturi, Miro

    2007-12-01

    Protein-kinase inhibitors are among the most advanced compounds in development using the new drug discovery paradigm of developing small-molecule drugs against specific molecular targets in cancer. After treatment with a cyclin dependent kinase CDK2 inhibitor in monkey, histopathological analysis of the eye showed specific cellular damage in the photoreceptor layer. Since this CDK2 inhibitor showed activity also on other CDKs, in order to investigate the mechanism of toxicity of this compound, we isolated cones and rods from the retina of normal monkey and humans by Laser Capture Microdissection. Using Real-Time PCR we first measured the expression of cyclin dependent protein-kinases (CDK)1, 2, 4, 5, Glycogen synthase kinase 3beta (GSK3beta) and microtubule associated protein TAU. We additionally verified the presence of these proteins in monkey eye sections by immuno-histochemistry and immunofluorescence analysis and afterwards quantified GSK3beta, phospho-GSK3beta and TAU by Reverse Phase Protein Microarrays. With this work we demonstrate how complementary gene expression and protein-based technologies constitute a powerful tool for the understanding of the molecular mechanism of a CDK2 inhibitor induced toxicity. Moreover, this investigative approach is helpful to better understand and characterize the mechanism of species-specific toxicities and further support a rational, molecular mechanism-based safety assessment in humans.

  15. Src kinases are required for a balanced production of IL-12/IL-23 in human dendritic cells activated by Toll-like receptor agonists.

    Directory of Open Access Journals (Sweden)

    Mirela Kuka

    Full Text Available BACKGROUND: Pathogen recognition by dendritic cells (DC is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation. It was previously shown by our group that Src kinases play a key role in cytokines production during TLR4 activation in human DC. PRINCIPAL FINDINGS: In this work we investigated the role of Src kinases during different TLRs triggering in human monocyte-derived DC (MoDC. We found that Src family kinases are important for a balanced production of inflammatory cytokines by human MoDC upon stimulation of TLR3 and 8 with their respective agonists. Disruption of this equilibrium through pharmacological inhibition of Src kinases alters the DC maturation pattern. In particular, while expression of IL-12 and other inflammatory cytokines depend on Src kinases, the induction of IL-23 and co-stimulatory molecules do not. Accordingly, DC treated with Src inhibitors are not compromised in their ability to induce CD4 T cell proliferation and to promote the Th17 subset survival but are less efficient in inducing Th1 differentiation. CONCLUSIONS: We suggest that the pharmacological modulation of DC maturation has the potential to shape the quality of the adaptive immune response and could be exploited for the treatment of inflammation-related diseases.

  16. Intermittent Administration of Parathyroid Hormone 1–34 Enhances Osteogenesis of Human Mesenchymal Stem Cells by Regulating Protein Kinase

    Directory of Open Access Journals (Sweden)

    Shu-Wen Kuo

    2017-10-01

    Full Text Available Human mesenchymal stem cells (hMSCs can differentiate into osteoblasts and are regulated by chemical cues. The recombinant N-terminal (1–34 amino acids fragment of the parathyroid hormone (PTH (1–34 is identified to promote osteogenesis. The osteoanabolic effects of intermittent PTH (1–34 treatment are linked to a complex consisting of signaling pathways; additionally, protein kinase C (PKC act as mediators of multifunctional signaling transduction pathways, but the role of PKC δ (PKCδ, a downstream target in regulating osteoblast differentiation during intermittent administration of PTH (1–34 is less studied and still remains elusive. The purpose of this study is to examine the role of PKCδ during intermittent and continuous PTH (1–34 administration using osteoblast-lineage-committed hMSCs. Relative gene expression of osteoblast-specific genes demonstrated significant upregulation of RUNX2, type I Collagen, ALP, and Osterix and increased alkaline phosphatase activity in the presence of PTH (1–34. Intermittent PTH (1–34 administration increased PKC activity at day 7 of osteogenic differentiation, whereas inhibition of PKC activity attenuated these effects. In addition, the specific isoform PKCδ was activated upon treatment. These findings demonstrate that intermittent PTH (1–34 treatment enhances the osteogenesis of hMSCs by upregulating osteoblast-specific genes via PKCδ activation.

  17. The cyclin-dependent kinase inhibitor flavopiridol sensitizes human hepatocellular carcinoma cells to TRAIL-induced apoptosis.

    Science.gov (United States)

    Miyashita, Kazumi; Shiraki, Katsuya; Fuke, Hiroyuki; Inoue, Tomoko; Yamanaka, Yutaka; Yamaguchi, Yumi; Yamamoto, Norihiko; Ito, Keiichi; Sugimoto, Kazushi; Nakano, Takeshi

    2006-08-01

    Flavopiridol was one of the first cyclin-dependent kinase inhibitors demonstrated to have an antitumor effect in several cancer types. Here, we investigated the effects of flavopiridol on TNF-related apoptosis-inducing ligand (TRAIL) in the human hepatocellular carcinoma (HCC) cell lines HLE and HepG2, and evaluated the role of flavopiridol in apoptosis. To better understand the mechanism of increased TRAIL sensitivity in HCC cells, we determined the effect of flavopiridol on cell surface expression of TRAIL and TRAIL receptors using flow cytometry analysis. The levels of survivin, FLIP, Bcl-xL and X-chromosome-linked IAP (XIAP) in treated and untreated cells was also determined. Flavopiridol decreased cell viability in a dose-dependent manner in the two HCC cell lines tested. The pan-caspase inhibitor z-VAD-FMK did not inhibit the effect. However, subtoxic levels of flavopiridol dramatically enhanced TRAIL-induced apoptosis in both cells. Flavopiridol up-regulated TRAIL, TRAIL-R1 and TRAIL-R2 in both cell lines. In addition, flavopiridol down-regulated expression of survivin in both cell lines, and expression of FLIP and Bcl-xL were down-regulated in HLE cells. In summary, flavopiridol augmented TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of survivin, FLIP and Bcl-xL. Thus, combining flavopiridol with a TRAIL agonist may prove to be an effective new strategy for treatment of HCC.

  18. Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

    Science.gov (United States)

    Ito, Mai; Arakawa, Toshiya; Okayama, Miki; Shitara, Akiko; Mizoguchi, Itaru; Takuma, Taishin

    2014-11-01

    The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement. © 2013 Wiley Publishing Asia Pty Ltd.

  19. Sequential Events in the Irreversible Thermal Denaturation of Human Brain-Type Creatine Kinase by Spectroscopic Methods

    Directory of Open Access Journals (Sweden)

    Yan-Song Gao

    2010-06-01

    Full Text Available The non-cooperative or sequential events which occur during protein thermal denaturation are closely correlated with protein folding, stability, and physiological functions. In this research, the sequential events of human brain-type creatine kinase (hBBCK thermal denaturation were studied by differential scanning calorimetry (DSC, CD, and intrinsic fluorescence spectroscopy. DSC experiments revealed that the thermal denaturation of hBBCK was calorimetrically irreversible. The existence of several endothermic peaks suggested that the denaturation involved stepwise conformational changes, which were further verified by the discrepancy in the transition curves obtained from various spectroscopic probes. During heating, the disruption of the active site structure occurred prior to the secondary and tertiary structural changes. The thermal unfolding and aggregation of hBBCK was found to occur through sequential events. This is quite different from that of muscle-type CK (MMCK. The results herein suggest that BBCK and MMCK undergo quite dissimilar thermal unfolding pathways, although they are highly conserved in the primary and tertiary structures. A minor difference in structure might endow the isoenzymes dissimilar local stabilities in structure, which further contribute to isoenzyme-specific thermal stabilities.

  20. Structural Injury after Lithium Treatment in Human and Rat Kidney involves Glycogen Synthase Kinase-3β Positive Epithelium

    DEFF Research Database (Denmark)

    Kjærsgaard, Gitte; Madsen, Kirsten; Marcussen, Niels

    2011-01-01

    Lithium is reabsorbed by distal nephron segments in sodium depleted states. It was hypothesized that lithium causes permanent injury to the developing kidney particularly in the sodium-retaining phase around weaning through entry into epithelial cells of the distal nephron and inhibition of glyco......Lithium is reabsorbed by distal nephron segments in sodium depleted states. It was hypothesized that lithium causes permanent injury to the developing kidney particularly in the sodium-retaining phase around weaning through entry into epithelial cells of the distal nephron and inhibition...... of glycogen synthase kinase-3β (GSK-3β). GSK-3β and pGSK-3β was investigated in a developing series of rat kidney cortex and medulla. Li+ was given to female wistar rats with litters through food pellets at postnatal (P) days 7-28. In human fetal and adult kidney the expression of GSK-3β was examined and also....... Lithium causes proliferation, structural injury and increases inactive pGSK-3β abundance in these segments. The data are compatible with epithelial entry of lithium and a causal role for GSK-3β in postnatal developing cortical collecting duct epithelium....

  1. 5′-AMP-activated Protein Kinase (AMPK) Supports the Growth of Aggressive Experimental Human Breast Cancer Tumors*

    Science.gov (United States)

    Laderoute, Keith R.; Calaoagan, Joy M.; Chao, Wan-ru; Dinh, Dominc; Denko, Nicholas; Duellman, Sarah; Kalra, Jessica; Liu, Xiaohe; Papandreou, Ioanna; Sambucetti, Lidia; Boros, Laszlo G.

    2014-01-01

    Rapid tumor growth can establish metabolically stressed microenvironments that activate 5′-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [13C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism. PMID:24993821

  2. 5'-AMP-activated protein kinase (AMPK) supports the growth of aggressive experimental human breast cancer tumors.

    Science.gov (United States)

    Laderoute, Keith R; Calaoagan, Joy M; Chao, Wan-ru; Dinh, Dominc; Denko, Nicholas; Duellman, Sarah; Kalra, Jessica; Liu, Xiaohe; Papandreou, Ioanna; Sambucetti, Lidia; Boros, Laszlo G

    2014-08-15

    Rapid tumor growth can establish metabolically stressed microenvironments that activate 5'-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [(13)C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. A Pan-BCL2 inhibitor renders bone-marrow-resident human leukemia stem cells sensitive to tyrosine kinase inhibition.

    Science.gov (United States)

    Goff, Daniel J; Court Recart, Angela; Sadarangani, Anil; Chun, Hye-Jung; Barrett, Christian L; Krajewska, Maryla; Leu, Heather; Low-Marchelli, Janine; Ma, Wenxue; Shih, Alice Y; Wei, Jun; Zhai, Dayong; Geron, Ifat; Pu, Minya; Bao, Lei; Chuang, Ryan; Balaian, Larisa; Gotlib, Jason; Minden, Mark; Martinelli, Giovanni; Rusert, Jessica; Dao, Kim-Hien; Shazand, Kamran; Wentworth, Peggy; Smith, Kristen M; Jamieson, Christina A M; Morris, Sheldon R; Messer, Karen; Goldstein, Lawrence S B; Hudson, Thomas J; Marra, Marco; Frazer, Kelly A; Pellecchia, Maurizio; Reed, John C; Jamieson, Catriona H M

    2013-03-07

    Leukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Retinoic acids and trichostatin A (TSA), a histone deacetylase inhibitor, induce human pyruvate dehydrogenase kinase 4 (PDK4) gene expression.

    Science.gov (United States)

    Kwon, Hye-Sook; Huang, Boli; Ho Jeoung, Nam; Wu, Pengfei; Steussy, Calvin N; Harris, Robert A

    2006-01-01

    Induction of pyruvate dehydrogenase kinase 4 (PDK4) conserves glucose and substrates for gluconeogenesis and thereby helps regulate blood glucose levels during starvation. We report here that retinoic acids (RA) as well as Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), regulate PDK4 gene expression. Two retinoic acid response elements (RAREs) to which retinoid X receptor alpha (RXRalpha) and retinoic acid receptor alpha (RARalpha) bind and activate transcription are present in the human PDK4 (hPDK4) proximal promoter. Sp1 and CCAAT box binding factor (CBF) bind to the region between two RAREs. Mutation of either the Sp1 or the CBF site significantly decreases basal expression, transactivation by RXRalpha/RARalpha/RA, and the ability of TSA to stimulate hPDK4 gene transcription. By the chromatin immunoprecipitation assay, RA and TSA increase acetylation of histones bound to the proximal promoter as well as occupancy of CBP and Sp1. Interaction of p300/CBP with E1A completely prevented hPDK4 gene activation by RXRalpha/RARalpha/RA and TSA. The p300/CBP may enhance acetylation of histones bound to the hPDK4 promoter and cooperate with Sp1 and CBF to stimulate transcription of the hPDK4 gene in response to RA and TSA.

  5. AKT serine/threonine protein kinase modulates baicalin-triggered autophagy in human bladder cancer T24 cells.

    Science.gov (United States)

    Lin, Chingju; Tsai, Shih-Chang; Tseng, Michael T; Peng, Shu-Fen; Kuo, Sheng-Chu; Lin, Meng-Wei; Hsu, Yuan-Man; Lee, Miau-Rong; Amagaya, Sakae; Huang, Wen-Wen; Wu, Tian-Shung; Yang, Jai-Sing

    2013-03-01

    Baicalin is one of the major compounds in the traditional Chinese medicinal herb from Scutellaria baicalensis Georgi. We investigated the molecular mechanisms of cell autophagy induced by baicalin in human bladder cancer T24 cells. Baicalin inhibited cell survival as shown by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. Baicalin did not induce apoptotic cell death in T24 cells by TUNEL and caspase-3 activity assay. Baicalin induced the acidic vesicular organelle cell autophagy marker, manifested by acridine orange (AO) and monodansylcadaverine (MDC) staining and cleavage of microtubule-associated protein 1 light chain 3 (LC3). The protein expression levels of the Atg 5, Atg 7, Atg 12, Beclin-1 and LC3-II were upregulated in T24 cells after baicalin treatment. Inhibition of autophagy by 3-methyl-adenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) reduced the cleavage of LC3 in T24 cells after baicalin treatment. Furthermore, protein expression levels of phospho-AKT (Ser473) and enzyme activity of AKT were downregulated in T24 cells after baicalin treatment. In conclusion, baicalin triggered cell autophagy through the AKT signaling pathway in T24 cells.

  6. Signaling via class IA Phosphoinositide 3-kinases (PI3K in human, breast-derived cell lines.

    Directory of Open Access Journals (Sweden)

    Veronique Juvin

    Full Text Available We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K in human breast-derived MCF10a (and iso-genetic derivatives and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5-trisphosphate (PtdIns(3,4,5P3 that can activate effectors, eg protein kinase B (PKB, and responses, eg migration. The PtdIns(3,4,5P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K, basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/- cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN(-/- MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely

  7. Downregulation of tyrosine threonine kinase inhibits tumor growth via G2/M arrest in human endometrioid endometrial adenocarcinoma.

    Science.gov (United States)

    Zhang, Jiamiao; Jiang, Yan; Zhao, Yu; Wang, Wanxue; Xie, Yiran; Wang, Huating; Yang, Yihua

    2017-07-01

    Endometrial cancer is the most common gynecologic malignancy, about 80% of which is endometrial endometrioid carcinoma. Dysregulation of spindle assembly checkpoint plays a vital role in endometrial endometrioid carcinoma tumorigenesis and progression. The purpose of this study was to explore how tyrosine threonine kinase, a spindle assembly checkpoint-related protein, promotes the endometrial endometrioid carcinoma progression. We found that both messenger RNA and protein levels of tyrosine threonine kinase in endometrial endometrioid carcinoma tissues are higher than those in normal endometrial tissues, and its expression is associated with tumor stages. Genetic depletion of tyrosine threonine kinase by RNA interference in two endometrial endometrioid carcinoma cell lines significantly inhibits cell proliferation and induces apoptosis. Mechanistically, depletion of tyrosine threonine kinase induces G2/M cell cycle arrest and triggers caspase-dependent cell apoptosis. Collectively, tyrosine threonine kinase is significantly upregulated in endometrial endometrioid carcinoma, and downregulation of tyrosine threonine kinase can suppress endometrial endometrioid carcinoma cell proliferation and promote apoptosis via G2/M cell cycle arrest. Our study demonstrates that tyrosine threonine kinase can be a potential therapeutic target for endometrial endometrioid carcinoma treatment.

  8. Apple polyphenols affect protein kinase C activity and the onset of apoptosis in human colon carcinoma cells.

    Science.gov (United States)

    Kern, Melanie; Pahlke, Gudrun; Balavenkatraman, Kamal Kumar; Böhmer, Frank D; Marko, Doris

    2007-06-27

    Polyphenol-rich apple extracts have been reported to suppress human colon cancer cell growth in vitro. The protein kinase C (PKC) is among the signaling elements known to play an important role in colon carcinogenesis. In the present study, we investigated whether apple polyphenols affect PKC activity and induce apoptosis in the human colon carcinoma cell line HT29. A polyphenol-rich apple juice extract (AE02) was shown to inhibit cytosolic PKC activity in a cell-free system. In contrast, incubation of HT29 cells for 1 or 3 h with AE02 up to 2 mg/mL did not affect the cytosolic PKC activity. After prolonged incubation (24 h), cytosolic PKC activity was modulated, albeit a u-shaped curve of effectiveness was observed, with an initial inhibitory effect followed by the recurrence and even induction of enzyme activity. Concomitantly, in the cytosol, a significant decrease of the protein levels of PKCalpha, PKCbetaII, and PKCgamma together with a significant increase of a proapoptotic PKCdelta fragment was observed. However, the effects on the protein levels of these PKC isoforms in the cytosol were not associated with translocation between the different cellular compartments but might instead result from the onset of apoptosis. Indeed, the treatment with AE02 was shown to induce apoptosis by the activation of caspase-3, DNA fragmentation, and cleavage of poly(ADP ribose) polymerase. So far, identified and available constituents of the apple extract did not contribute substantially to the observed effects on PKC and apoptosis induction. In summary, apple polyphenols were found to inhibit PKC activity in a cell-free system. However, our results indicate that within intact cells PKC does not represent the primary target of apple polyphenols but appears to be affected in the course of apoptosis induction.

  9. Creatine kinase rate constant in the human heart measured with 3D‐localization at 7 tesla

    Science.gov (United States)

    Robson, Matthew D.; Neubauer, Stefan; Rodgers, Christopher T.

    2016-01-01

    Purpose We present a new Bloch‐Siegert four Angle Saturation Transfer (BOAST) method for measuring the creatine kinase (CK) first‐order effective rate constant kf in human myocardium at 7 tesla (T). BOAST combines a variant of the four‐angle saturation transfer (FAST) method using amplitude‐modulated radiofrequency pulses, phosphorus Bloch‐Siegert B1+‐mapping to determine the per‐voxel flip angles, and nonlinear fitting to Bloch simulations for postprocessing. Methods Optimal flip angles and repetition time parameters were determined from Monte Carlo simulations. BOAST was validated in the calf muscle of two volunteers at 3T and 7T. The myocardial CK forward rate constant was then measured in 10 volunteers at 7T in 82 min (after 1H localization). Results BOAST kfCK values were 0.281 ± 0.002 s−1 in the calf and 0.35 ± 0.05 s−1 in myocardium. These are consistent with literature values from lower fields. Using a literature values for adenosine triphosphate concentration, we computed CK flux values of 4.55 ± 1.52 mmol kg−1 s−1. The sensitive volume for BOAST depends on the B1 inhomogeneity of the transmit coil. Conclusion BOAST enables measurement of the CK rate constant in the human heart at 7T, with spatial localization in three dimensions to 5.6 mL voxels, using a 10‐cm loop coil. Magn Reson Med 78:20–32, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PMID:27579566

  10. pH-dependent relationship between thermodynamic and kinetic stability in the denaturation of human phosphoglycerate kinase 1.

    Science.gov (United States)

    Pey, Angel L

    2014-08-01

    Human phosphoglycerate kinase 1 (hPGK1) is a glycolytic enzyme essential for ATP synthesis, and it is implicated in different pathological conditions such as inherited diseases, oncogenesis and activation of drugs for cancer and viral treatments. Particularly, mutations in hPGK1 cause human PGK1 deficiency, a rate metabolic conformational disease. We have recently found that most of these mutations cause protein kinetic destabilization by significant changes in the structure/energetics of the transition state for irreversible denaturation. In this work, we explore the relationships between protein conformation, thermodynamic and kinetic stability in hPGK1 by performing comprehensive analyses in a wide pH range (2.5-8). hPGK1 remains in a native conformation at pH 5-8, but undergoes a conformational transition to a molten globule-like state at acidic pH. Interestingly, hPGK1 kinetic stability remains essentially constant at pH 6-8, but is significantly reduced when pH is decreased from 6 to 5. We found that this decrease in kinetic stability is caused by significant changes in the energetic/structural balance of the denaturation transition state, which diverge from those found for disease-causing mutations. We also show that protein kinetic destabilization by acidic pH is strongly linked to lower thermodynamic stability, while in disease-causing mutations seems to be linked to lower unfolding cooperativity. These results highlight the plasticity of the hPGK1 denaturation mechanism that responds differently to changes in pH and in disease-causing mutations. New insight is presented into the different factors contributing to hPGK1 thermodynamic and kinetic stability and the role of denaturation mechanisms in hPGK1 deficiency. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  11. Novel covalent modification of human anaplastic lymphoma kinase (ALK) and potentiation of crizotinib-mediated inhibition of ALK activity by BNP7787.

    Science.gov (United States)

    Parker, Aulma R; Petluru, Pavankumar N; Nienaber, Vicki L; Zhao, Min; Ayala, Philippe Y; Badger, John; Chie-Leon, Barbara; Sridhar, Vandana; Logan, Cheyenne; Kochat, Harry; Hausheer, Frederick H

    2015-01-01

    BNP7787 (Tavocept, disodium 2,2'-dithio-bis-ethanesulfonate) is a novel, investigational, water-soluble disulfide that is well-tolerated and nontoxic. In separate randomized multicenter Phase II and Phase III clinical trials in non-small-cell lung cancer (NSCLC) patients, treatment with BNP7787 in combination with standard chemotherapy resulted in substantial increases in the overall survival of patients with advanced adenocarcinoma of the lung in the first-line treatment setting. We hypothesized that BNP7787 might interact with and modify human anaplastic lymphoma kinase (ALK). At least seven different variants of ALK fusions with the gene encoding the echinoderm microtubule-associated protein-like 4 (EML4) are known to occur in NSCLC. EML4-ALK fusions are thought to account for approximately 3% of NSCLC cases. Herein, we report the covalent modification of the kinase domain of human ALK by a BNP7787-derived mesna moiety and the functional consequences of this modification in ALK assays evaluating kinase activity. The kinase domain of the ALK protein crystallizes as a monomer, and BNP7787-derived mesna-cysteine adducts were observed at Cys 1235 and Cys 1156. The BNP7787-derived mesna adduct at Cys 1156 is located in close proximity to the active site and results in substantial disorder of the P-loop and activation loop (A-loop). Comparison with the P-loop of apo-ALK suggests that the BNP7787-derived mesna adduct at Cys 1156 interferes with the positioning of Phe 1127 into a small pocket now occupied by mesna, resulting in a destabilization of the loop's binding orientation. Additionally, in vitro kinase activity assays indicate that BNP7787 inhibits ALK catalytic activity and potentiates the activity of the ALK-targeted drug crizotinib.

  12. Virulence factors of Staphylococcus aureus induce Erk-MAP kinase activation and c-Fos expression in S9 and 16HBE14o- human airway epithelial cells.

    Science.gov (United States)

    Below, Sabine; Konkel, Anne; Zeeck, Cathrin; Müller, Christian; Kohler, Christian; Engelmann, Susanne; Hildebrandt, Jan-Peter

    2009-03-01

    Part of the innate defense of bronchial epithelia against bacterial colonization is regulated secretion of salt, water, and mucus as well as defensins and cytokines involving MAP kinase activation and alterations in early gene expression. We tested two different types of immortalized human airway epithelial cells (S9, 16HBE14o-) for activation of Erk-type MAP kinases and for expression of c-Fos on treatment with Staphylococcus aureus culture supernatants from the stationary growth phase [optical density (OD)(540 nm) = 10] or with recombinant S. aureus hemolysins A and B (Hla, Hlb). OD10 supernatants activated Erk-type MAP kinases and c-Fos expression in a concentration-dependent manner. Hla induced Erk-type kinase phosphorylation in S9 but not in 16HBE14o- cells. Hlb induced Erk activation in either cell type. Basal and stimulated levels of Erk-type MAP kinase phosphorylation were sensitive to the Mek1 inhibitor PD-98059, indicating that the bacterial products activated the entire signaling cascade that coregulates IL-8 induction and secretion. While c-Fos expression was enhanced by OD10 supernatants, Hla, and Hlb in S9 cells, 16HBE14o- cells responded to OD10 supernatant and Hlb but not to Hla. In S9 cells, PD-98059 suppressed c-Fos upregulation by OD10 supernatant, Hla, or Hlb, indicating that c-Fos expression requires activation of Erk-type MAP kinases. In 16HBE14o- cells, however, c-Fos expression by OD10 supernatant was sensitive to PD-98059, while that induced by Hlb was not. This indicates that ingredients of OD10 supernatants other than Hla or Hlb are activating Erk-type MAP kinases in 16HBE14o- cells and that other intracellular signaling systems apart from Erk-type MAP kinases contribute to Hlb-mediated regulation of c-Fos. Thus interaction of bacterial factors with airway epithelial cells may be highly cell type specific.

  13. [Therapeutic effect of flavopiridol, a small molecular cyclin-dependent kinase inhibitor, in human ovarian carcinoma].

    Science.gov (United States)

    Song, Yue; Shen, Keng; Tang, Ping-ping

    2007-11-01

    To investigate the antitumor effect of flavopiridol in ovarian cancer. After the treatment with flavopiridol of AO cells, cell apoptotic rate and cell cycle distribution were detected by flow cytometer and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL). Real time PCR was used to detect the expression of cyclin D and active caspase-3 in AO cells. Subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma using AO cells in BALB/c nude mice were established. The mouse survival rates were measured for abdominally spread tumor models and the volume of tumor nodules was determined for subcutaneous tumor models following the treatments of flavopiridol. TUNEL was used to detect cell apoptosis, and immunohistochemistry was used to measure microvessel density (MVD) in tumor tissues. AO cells showed apoptotic rates of 4.1%, 10.7% and 7.6% following the treatments with flavopiridol at 150, 300 and 500 nmol/L respectively, accompanied by an increase in G(1) progression and a decrease in S phase progression. The level of active caspase-3 increased (2.55 vs 2.49) and the level of cyclin D expression decreased significantly (0.25 vs 0.69, P flavopiridol. Flavopiridol prolonged mouse survival [mean survival time of (141 +/- 14) days] and suppressed tumor growth significantly (tumor growth suppression rate of 40%), when compared with treatment using phosphate-buffered saline [(106 +/- 11) days, P flavopiridol. MVD of tumor tissue was 12 +/- 5 following flavopiridol treatment, significantly higher than that of 35 +/- 10 treated with phosphate-buffered saline (P Flavopiridol results in significant suppression of ovarian carcinoma cell growth and prolongs survival of mice.

  14. Subcellular proteomic approach for identifying the signaling effectors of protein kinase C-?2 under high glucose conditions in human umbilical vein endothelial cells

    OpenAIRE

    Zhang, Min; Sun, Fang; Chen, Fangfang; Zhou, Bo; DUAN, YAQIAN; SU, HONG; LIN, XUEBO

    2015-01-01

    The high glucose-induced activation of protein kinase C-?2 (PKC-?2) has an essential role in the pathophysiology of diabetes-associated vascular disease. In the present study, human umbilical vein endothelial cells (HUVECs) were cultured in high and normal glucose conditions prior to being infected with a recombinant adenovirus to induce the overexpression of PKC-?2. The activity of PKC-?2 was also decreased using a selective PKC-?2 inhibitor. A series of two-dimensional electrophoresis image...

  15. Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells

    OpenAIRE

    Guon, Tae Eun; Chung, Ha Sook

    2017-01-01

    The present study was performed to determine the effect of Moringa oleifera fruit extract on the apoptosis of human melanoma A2058 cells. A2058 cells were treated for 72 h with Moringa oleifera fruit extract at 50–100 µg/ml, and cell viability with apoptotic changes was examined. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) was examined. It was revealed that Moringa oleifera fruit extract significantly inhibited the cell viability and promoted...

  16. axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase

    Energy Technology Data Exchange (ETDEWEB)

    O' Bryan, J.P.; Frye, R.A.; Cogswell, P.C.; Neubauer, A.; Kitch, B.; Prokop, C.; Earp, H.S.; Liu, E.T. (Univ. of North Carolina, Chapel Hill (United States)); Espinosa, R. III; Le Beau, M.M. (Univ. of Chicago, IL (United States))

    1991-10-01

    Using a sensitive transfection-tumorigenicity assay, the authors have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antophosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type II and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.

  17. Interferon-mediated antiviral state in human MRC5 cells in the absence of detectable levels of 2-5A synthetase and protein kinase.

    Science.gov (United States)

    Meurs, E; Hovanessian, A G; Montagnier, L

    1981-02-01

    Treatment of human HeLa and MRC5 cells with human alpha (leukocyte) and beta (fibroblast) interferon results in the development of an antiviral state against two types of viruses: vesicular stomatitis virus (rhabdovirus) and encephalomyocarditis virus (picornavirus). These cells, however, differ in their ability to synthesize the two double-stranded (ds) RNA-dependent enzymatic activities, pppA(2'p5'A)n synthetase (2-5A synthetase) and protein kinase which have been reported to be induced in several cell lines by interferon. Both the 2-5A synthetase and the protein kinase are enhanced by several fold in HeLa cells on treatment with interferon. In contrast, neither the 2-5A synthetase nor the protein kinase can be detected in MRC5 cell treated or not treated with interferon. The lack of detection of the 2-5A synthetase in MRC5 cells is not associated with the absence of the other components of the 2-5A system (2-5A dependent nuclease and 2'-phosphodiesterase). We have previously shown that MRC5 cells are sensitive to the action of 2-5A and furthermore the inhibitory action of 2-5A on these cells is transient. Mixing experiments between HeLa and MRC5 cell fractions after partial purification on columns of poly(I).poly(C)-Sepharose, showed that the absence of detection of the protein kinase activity in MRC5 cells cannot be attributed to the presence of phosphatases or other inhibitors of phosphorylation in control or interferon-treated MRC5 cell extracts. In addition, we show that the interferon-mediated protein kinase activity in HeLa cell extracts can be precipitated by treatment at pH 5, a procedure which leads to an enhanced level of detectable protein kinase activity in general. Once again, however, MRC5 cell extracts fail to show any interferon-mediated protein kinase activity. These results suggest that either the two enzyme activities are not necessary for the development of the antiviral response induced by interferon or the intracellular events leading to

  18. Direct association of heat shock protein 20 (HSPB6 with phosphoinositide 3-kinase (PI3K in human hepatocellular carcinoma: regulation of the PI3K activity.

    Directory of Open Access Journals (Sweden)

    Rie Matsushima-Nishiwaki

    Full Text Available HSP20 (HSPB6, one of small heat shock proteins (HSPs, is constitutively expressed in various tissues and has several functions. We previously reported that the expression levels of HSP20 in human hepatocellular carcinoma (HCC cells inversely correlated with the progression of HCC, and that HSP20 suppresses the growth of HCC cells via the AKT and mitogen-activated protein kinase signaling pathways. However, the exact mechanism underlying the effect of HSP20 on the regulation of these signaling pathways remains to be elucidated. To clarify the details of this effect in HCC, we explored the direct targets of HSP20 in HCC using human HCC-derived HuH7 cells with HSP20 overexpression. HSP20 proteins in the HuH7 cells were coimmunoprecipitated with the p85 regulatory subunit and p110 catalytic subunit of phosphoinositide 3-kinase (PI3K, an upstream kinase of AKT. Although HSP20 overexpression in HCC cells failed to affect the expression levels of PI3K, the activity of PI3K in the unstimulated cells and even in the transforming growth factor-α stimulated cells were downregulated by HSP20 overexpression. The association of HSP20 with PI3K was also observed in human HCC tissues in vivo. These findings strongly suggest that HSP20 directly associates with PI3K and suppresses its activity in HCC, resulting in the inhibition of the AKT pathway, and subsequently decreasing the growth of HCC.

  19. Escherichia coli-derived and Staphylococcus aureus-derived extracellular vesicles induce MUC5AC expression via extracellular signal related kinase 1/2 and p38 mitogen-activated protein kinase in human airway epithelial cells.

    Science.gov (United States)

    Bae, Chang Hoon; Choi, Yoon Seok; Song, Si-Youn; Kim, Yoon-Keun; Kim, Yong-Dae

    2017-01-01

    Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) release extracellular vesicles (EVs). E. coli-derived and S. aureus-derived EVs are associated with neutrophilic respiratory inflammation. In neutrophilic respiratory inflammation of human, expression of mucin is increased in airway epithelial cells and is associated with increased morbidity and mortality of the affected patients. However, no study on the effects of EVs on expression of mucin genes has been reported in airway epithelial cells. Therefore, this study was conducted in order to examine the effects and the brief signaling pathways of E. coli-derived and S. aureus-derived EVs on MUC5AC expression in human airway epithelial cells. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effects and signaling pathways of E. coli-derived and S. aureus-derived EVs on MUC5AC expression were examined using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). E. coli-derived and S. aureus-derived EVs induced MUC5AC expression. E. coli-derived and S. aureus-derived EVs significantly activated phosphorylation of extracellular signal related kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) and p38 MAPK. ERK1/2 MAPK inhibitor, p38 MAPK inhibitor, ERK1/2 MAPK siRNA, and p38 MAPK siRNA significantly blocked E. coli-derived and S. aureus-derived EVs induced MUC5AC messenger RNA (mRNA) expression. The results of this study suggest that E. coli-derived and S. aureus-derived EVs induced MUC5AC expression via ERK1/2 and p38 MAPK signaling pathways in human airway epithelial cells. © 2016 ARS-AAOA, LLC.

  20. Stimulation of extracellular signal-regulated kinases and proliferation in the human gastric cancer cells KATO-III by obestatin.

    Science.gov (United States)

    Pazos, Yolanda; Alvarez, Carlos J P; Camiña, Jesus P; Casanueva, Felipe F

    2007-12-01

    Obestatin, the ghrelin-associated peptide, activates cell proliferation in the gastric cancer cell line KATO-III. The results showed that this peptide induced cell proliferation by mitogen-activated kinase kinase/extracellular signal-regulated kinases1/2 (ERK1/2) phosphorylation. A sequential analysis of the obestatin transmembrane signalling pathway indicated that the ERK1/2 activity is partially blocked after preincubation of the cells with pertussis toxin, as well as by wortmannin (an inhibitor of phosphoinositide 3-kinase (PI3K)), staurosporine (an inhibitor of protein kinase C (PKC)) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2, which inhibits the non receptor tyrosine kinase Src). Upon administration of obestatin, the intracellular levels of phospho-PKCepsilon- and theta-isoenzymes rise with similar time-courses, from which PKCepsilon appears to be the responsible for ERK1/2 response. Based on the experimental data, a signalling pathway involving the consecutive activation of G(i), PI3K, novel PKCepsilon and Src for ERK1/2 activation is proposed. These results point to a functionally active peptide that regulates proliferation of the gastric cancer cells KATO-III.

  1. Gene duplications and losses among vertebrate deoxyribonucleoside kinases of the non-TK1 Family

    DEFF Research Database (Denmark)

    Mutahir, Zeeshan; Christiansen, Louise Slot; Clausen, Anders R.

    2016-01-01

    Deoxyribonucleoside kinases (dNKs) salvage deoxyribonucleosides (dNs) and catalyze the rate limiting step of this salvage pathway by converting dNs into corresponding monophosphate forms. These enzymes serve as an excellent model to study duplicated genes and their evolutionary history. So far......, among vertebrates only four mammalian dNKs have been studied for their substrate specificity and kinetic properties. However, some vertebrates, such as fish, frogs, and birds, apparently possess a duplicated homolog of deoxycytidine kinase (dCK). In this study, we characterized a family of d......CK/deoxyguanosine kinase (dGK)-like enzymes from a frog Xenopus laevis and a bird Gallus gallus. We showed that X. laevis has a duplicated dCK gene and a dGK gene, whereas G. gallus has a duplicated dCK gene but has lost the dGK gene. We cloned, expressed, purified, and subsequently determined the kinetic parameters...

  2. Effects of combined therapy of Phosphatidylinositol 3p-Kinase and Paclitaxel in human lung cancer nude mice model

    Directory of Open Access Journals (Sweden)

    Yajuan REN

    2008-04-01

    Full Text Available Background and objective Increasing evidence suggests that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer therapy. The aims of this study is to evaluate the effects of combined therapy of Phosphatidylinositol 3-Kinase inhibitor(LY294002 and Paclitaxel in athymic mice xenogeneic transplant model of lung cancer and to reveal the possible mechanisms involved.Methods Eighteen athymic mice were randomly divided into 3 groups (control, paclitaxel alone and paclitaxel plus LY294002, and were treated respectively. Athymic mice xenogeneic transplant model was establishedby inoculation (sc with A549 lung cancer cells. Body mass (BM and diameter of tumor mass were measured. Furthermore,the protein expressions of CyclinD1,bcl-2 and bax in tumor tissues were analyzed with immunohistochemistry. Results The tumor-inhibiting rate of paclitaxel plus LY294002 (92.47% was significantly higher than the paclitaxel alone (65.59%(P<0.05.The protein expression of bcl-2 in paclitaxel plus LY294002 group were significantly higher, while bax wassignificantly lower than that in the other two groups (P <0.05. The protein expression of CyclinD1 was significantly lower than the control group (P <0.05. Conclusion LY294002 can enhance the effects of paclitaxel in the treatment of lung cancer and CyclinD1, bcl-2 and bax may be involved in its inhibitory effects.

  3. Cyclooxygenase 2-dependent and independent activation of Akt through casein kinase 2α contributes to human bladder cancer cell survival

    Directory of Open Access Journals (Sweden)

    Fujimoto Kiyohide

    2011-05-01

    Full Text Available Abstract Background Survival rate for patients presenting muscle invasive bladder cancer is very low, and useful therapeutic target has not been identified yet. In the present study, new COX2 downstream signals involved in urothelial carcinoma cell survival were investigated in vitro and in vivo. Methods COX2 gene was silenced by siRNA transfection. Orthotopic implantation animal model and transurethral instillation of siRNA with atelocollagen was constructed to examine the effects of COX2 knockdown in vivo. Cell cycle was examined by flowcytoketry. Surgical specimens derived from patients with urinary bladder cancer (all were initially diagnosed cases were used for immunohistochemical analysis of the indicated protein expression in urothelial carcinoma cells. Results Treatment with the COX2 inhibitor or knockdown of COX2 reduced expression of casein kinase (CK 2 α, a phophorylated Akt and urokinase type plasminogen activator (uPA, resulting in p27 induction, cell cycle arrest at G1 phase and cell growth suppression in human urothelial carcinoma cell lines expressing COX2. Silencing of CK2α exhibited the similar effects. Even in UMUC3 cells lacking the COX2 gene, COX2 inhibition also inhibited cell growth through down-regulation of the CK2α-Akt/uPA axis. The mouse orthotropic bladder cancer model demonstrated that the COX2 inhibitor, meloxicam significantly reduced CK2α, phosphorylated Akt and uPA expression, whereas induced p27 by which growth and invasiveness of bladder cancer cells were strongly inhibited. Immunohistochemically, high expression of COX2, CK2α and phosphorylated form of Akt was found in high-grade, invasive carcinomas as well as carcinoma in situ, but not in low-grade and noninvasive phenotypes. Conclusions COX2-dependent and independent activation of CK2α-Akt/uPA signal is mainly involved in urothelial carcinoma cell survival, moreover, not only COX2 but also CK2α could be direct targets of COX2 inhibitors.

  4. Protein Kinase CK2 Regulates Cytoskeletal Reorganization during Ionizing Radiation-Induced Senescence of Human Mesenchymal Stem Cells

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    Wang, Daojing; Jang, Deok-Jin

    2009-08-21

    Human mesenchymal stem cells (hMSC) are critical for tissue regeneration. How hMSC respond to genotoxic stresses and potentially contribute to aging and cancer remain underexplored. We demonstrated that ionizing radiation induced cellular senescence of hMSC over a period of 10 days, showing a critical transition between day 3 and day 6. This was confirmed by senescence-associated beta-galactosidase (SA-{beta}-gal) staining, protein expression profiles of key cell cycle regulators (retinoblastoma (Rb) protein, p53, p21{sup waf1/Cip1}, and p16{sup INK4A}), and senescence-associated secretory phenotypes (SASPs) (IL-8, IL-12, GRO, and MDC). We observed dramatic cytoskeletal reorganization of hMSC through reduction of myosin-10, redistribution of myosin-9, and secretion of profilin-1. Using a SILAC-based phosphoproteomics method, we detected significant reduction of myosin-9 phosphorylation at Ser1943, coinciding with its redistribution. Importantly, through treatment with cell permeable inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT)), and gene knockdown using RNA interference, we identified CK2, a kinase responsible for myosin-9 phosphorylation at Ser1943, as a key factor contributing to the radiation-induced senescence of hMSC. We showed that individual knockdown of CK2 catalytic subunits CK2{alpha} and CK2{alpha}{prime} induced hMSC senescence. However, only knockdown of CK2{alpha} resulted in morphological phenotypes resembling those of radiation-induced senescence. These results suggest that CK2{alpha} and CK2{alpha}{prime} play differential roles in hMSC senescence progression, and their relative expression might represent a novel regulatory mechanism for CK2 activity.

  5. Soluble human leukocyte antigen-g5 activates extracellular signal-regulated protein kinase signaling and stimulates trophoblast invasion.

    Science.gov (United States)

    Guo, YiFan; Lee, Cheuk-Lun; So, Kam-Hei; Gao, Jing; Yeung, William S B; Yao, YuanQing; Lee, Kai-Fai

    2013-01-01

    Soluble human leukocyte antigen-G (HLA-G) is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and activation of extracellular signal-regulated protein kinase (ERK) signaling pathway. Recombinant HLA-G5 protein over-expressed in E. coli BL21 was purified to near homogeneity. We studied the expression of HLA-G5 and its receptors, the leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), in primary trophoblasts and trophoblastic (JAr and JEG-3) cell lines by florescence-labeled HLA-G5. HLA-G5 was detected in the primary trophoblasts and JEG-3 cells. The LILRB1 and KIR2DL4 receptors were expressed in both primary trophoblasts and trophoblastic cell lines. HLA-G5 stimulated cell invasion (ptrophoblastic cells. HLA-G5 activated the ERK signaling pathway and induced ERK1/2 phosphorylation in the trophoblastic cell lines. Addition of ERK inhibitors (U0126 and PD98059) nullified the stimulatory effect of HLA-G5 on trophoblastic cell invasion. Taken together, HLA-G5 induced trophoblast invasion by binding to KIR2DL4 and LILRB1, by increasing uPA and MMPs expressions and by activating the ERK signaling pathway.

  6. Soluble human leukocyte antigen-g5 activates extracellular signal-regulated protein kinase signaling and stimulates trophoblast invasion.

    Directory of Open Access Journals (Sweden)

    YiFan Guo

    Full Text Available Soluble human leukocyte antigen-G (HLA-G is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and activation of extracellular signal-regulated protein kinase (ERK signaling pathway. Recombinant HLA-G5 protein over-expressed in E. coli BL21 was purified to near homogeneity. We studied the expression of HLA-G5 and its receptors, the leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1 and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4, in primary trophoblasts and trophoblastic (JAr and JEG-3 cell lines by florescence-labeled HLA-G5. HLA-G5 was detected in the primary trophoblasts and JEG-3 cells. The LILRB1 and KIR2DL4 receptors were expressed in both primary trophoblasts and trophoblastic cell lines. HLA-G5 stimulated cell invasion (p<0.05 and increased urokinase (uPA and matrix metalloproteinases (MMPs transcripts and their activity (p<0.05 in trophoblastic cells. HLA-G5 activated the ERK signaling pathway and induced ERK1/2 phosphorylation in the trophoblastic cell lines. Addition of ERK inhibitors (U0126 and PD98059 nullified the stimulatory effect of HLA-G5 on trophoblastic cell invasion. Taken together, HLA-G5 induced trophoblast invasion by binding to KIR2DL4 and LILRB1, by increasing uPA and MMPs expressions and by activating the ERK signaling pathway.

  7. Human Cytomegalovirus UL97 Kinase Activity Is Required for the Hyperphosphorylation of Retinoblastoma Protein and Inhibits the Formation of Nuclear Aggresomes

    Energy Technology Data Exchange (ETDEWEB)

    Prichard, Mark N.; Sztul, Elizabeth; Daily, Shannon L.; Perry, Amie L.; Frederick, Samuel L.; Gill, Rachel B.; Hartline, Caroll B.; Streblow, Daniel N.; Varnum, Susan M.; Smith, Richard D.; Kern, Earl R.

    2008-05-01

    Cells infected with human cytomegalovirus in the absence of UL97 kinase activity produce large nuclear aggregates that sequester considerable quantities of viral proteins. A transient expression assay suggested that pp71 and IE1 were also involved in this process, and this suggestion was significant, since both proteins have been reported to interact with components of promyelocytic leukemia (PML) bodies (ND10) and also interact functionally with retinoblastoma pocket proteins (RB). PML bodies have been linked to the formation of nuclear aggresomes, and colocalization studies suggested that viral proteins were recruited to these structures and that UL97 kinase activity inhibited their formation. Proteins associated with PML bodies were examined by Western blot analysis, and pUL97 appeared to specifically affect the phosphorylation of RB in a kinasedependent manner. Three consensus RB binding motifs were identified in the UL97 kinase, and recombinant viruses were constructed in which each was mutated to assess a potential role in the phosphorylation of RB and the inhibition of nuclear aggresome formation. The mutation of either the conserved LxCxE RB binding moti for the lysine required for kinase activity impaired the ability of the virus to stabilize and phosphorylate RB. We concluded from these studies that both UL97 kinase activity and the LxCxE RB binding motif are required for the phosphorylation and stabilization of RB in infected cells and that this effect can be antagonized by the antiviral drug maribavir. These data also suggest a potential link between RB function and the formation of aggresomes.

  8. Dual Inhibition of Topoisomerase II and Tyrosine Kinases by the Novel Bis-Fluoroquinolone Chalcone-Like Derivative HMNE3 in Human Pancreatic Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Yong-Chao Ma

    Full Text Available Both tyrosine kinase and topoisomerase II (TopII are important anticancer targets, and their respective inhibitors are widely used in cancer therapy. However, some combinations of anticancer drugs could exhibit mutually antagonistic actions and drug resistance, which further limit their therapeutic efficacy. Here, we report that HMNE3, a novel bis-fluoroquinolone chalcone-like derivative that targets both tyrosine kinase and TopII, induces tumor cell proliferation and growth inhibition. The viabilities of 6 different cancer cell lines treated with a range of HMNE3 doses were detected using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Cellular apoptosis was determined using Hoechst 33258 fluorescence staining and the terminal deoxynucleotidyl transferase (TdT dUTP nick-end labeling (TUNEL assay. The expression of activated Caspase-3 was examined by immunocytochemistry. The tyrosine kinase activity was measured with a human receptor tyrosine kinase (RTK detection kit using a horseradish peroxidase (HRP-conjugated phosphotyrosine (pY20 antibody as the substrate. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. The expression levels of the P53, Bax, Bcl-2, Caspase-3, -8, -9, p-cSrc, c-Src and topoisomerase II proteins were detected by western blot analysis. The proliferation of five of the six cancer cell lines was significantly inhibited by HMNE3 at 0.312 to 10 μmol/L in a time- and dose-dependent manner. Treatment of the Capan-1 and Panc-1 cells with 1.6 to 3.2 μM HMNE3 for 48 h significantly increased the percentage of apoptotic cells (P<0.05, and this effect was accompanied by a decrease in tyrosine kinase activity. HMNE3 potentially inhibited tyrosine kinase activity in vitro with an IC50 value of 0.64±0.34 μmol/L in Capan-1 cells and 3.1±0.86 μmol/L in Panc-1 cells. The activity of c-Src was significantly inhibited by HMNE3 in a dose

  9. Creatine kinase rate constant in the human heart measured with 3D-localization at 7 tesla.

    Science.gov (United States)

    Clarke, William T; Robson, Matthew D; Neubauer, Stefan; Rodgers, Christopher T

    2017-07-01

    We present a new Bloch-Siegert four Angle Saturation Transfer (BOAST) method for measuring the creatine kinase (CK) first-order effective rate constant kf in human myocardium at 7 tesla (T). BOAST combines a variant of the four-angle saturation transfer (FAST) method using amplitude-modulated radiofrequency pulses, phosphorus Bloch-Siegert B1+-mapping to determine the per-voxel flip angles, and nonlinear fitting to Bloch simulations for postprocessing. Optimal flip angles and repetition time parameters were determined from Monte Carlo simulations. BOAST was validated in the calf muscle of two volunteers at 3T and 7T. The myocardial CK forward rate constant was then measured in 10 volunteers at 7T in 82 min (after 1 H localization). BOAST kfCK values were 0.281 ± 0.002 s-1 in the calf and 0.35 ± 0.05 s-1 in myocardium. These are consistent with literature values from lower fields. Using a literature values for adenosine triphosphate concentration, we computed CK flux values of 4.55 ± 1.52 mmol kg-1 s-1 . The sensitive volume for BOAST depends on the B1 inhomogeneity of the transmit coil. BOAST enables measurement of the CK rate constant in the human heart at 7T, with spatial localization in three dimensions to 5.6 mL voxels, using a 10-cm loop coil. Magn Reson Med 78:20-32, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.

  10. c-Jun N-terminal kinase is required for thermotherapy-induced apoptosis in human gastric cancer cells.

    Science.gov (United States)

    Xiao, Feng; Liu, Bin; Zhu, Qing-Xian

    2012-12-28

    To investigate the role of c-Jun N-terminal kinase (JNK) in thermotherapy-induced apoptosis in human gastric cancer SGC-7901 cells. Human gastric cancer SGC-7901 cells were cultured in vitro. Following thermotherapy at 43°C for 0, 0.5, 1, 2 or 3 h, the cells were cultured for a further 24 h with or without the JNK specific inhibitor, SP600125 for 2 h. Apoptosis was evaluated by immunohistochemistry [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)] and flow cytometry (Annexin vs propidium iodide). Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The production of p-JNK, Bcl-2, Bax and caspase-3 proteins was evaluated by Western blotting. The expression of JNK at mRNA level was determined by reverse transcription polymerase chain reaction. The proliferation of gastric carcinoma SGC-7901 cells was significantly inhibited following thermotherapy, and was 32.7%, 30.6%, 43.8% and 52.9% at 0.5, 1, 2 and 3 h post-thermotherapy, respectively. Flow cytometry analysis revealed an increased population of SGC-790l cells in G0/G1 phase, but a reduced population in S phase following thermotherapy for 1 or 2 h, compared to untreated cells (P thermotherapy for 0.5, 1, 2 or 3 h, compared to the untreated group (46.5% ± 0.23%, 39.9% ± 0.53%, 56.6% ± 0.35% and 50.4% ± 0.29% vs 7.3% ± 0.10%, P thermotherapy, compared to mock-inhibitor treatment, which was in line with the decreased rate of apoptosis. The expression of Bcl-2 was consistent with thermotherapy alone. Thermotherapy induced apoptosis in gastric cancer cells by promoting p-JNK at the mRNA and protein levels, and up-regulated the expression of Bax and caspase-3 proteins. Bcl-2 may play a protective role during thermotherapy. Activation of JNK via the Bax-caspase-3 pathway may be important in thermotherapy-induced apoptosis in gastric cancer cells.

  11. Endothelial sphingosine kinase/SPNS2 axis is critical for vessel-like formation by human mesoangioblasts.

    Science.gov (United States)

    Laurenzana, Anna; Cencetti, Francesca; Serratì, Simona; Bruno, Gennaro; Japtok, Lukasz; Bianchini, Francesca; Torre, Eugenio; Fibbi, Gabriella; Del Rosso, Mario; Bruni, Paola; Donati, Chiara

    2015-10-01

    The interaction between endothelial cells and pericytes is crucial for the stabilization of newly formed vessels in angiogenesis. The comprehension of the mechanisms regulating pericyte recruitment might open therapeutical perspectives on vascular-related pathologies. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that derives from sphingomyelin catabolism and regulates biological functions in cell survival, proliferation, and differentiation. In this study, we aimed to identify the role of S1P axis in the intercellular communication between human mesenchymal progenitor mesoangioblasts (MAB) and endothelial cells (human microvascular endothelial cells (H-MVEC)) in the formation of capillary-like structures. We demonstrated that the S1P biosynthetic pathway brought about by sphingosine kinases (SK) SK1 and SK2 as well as spinster homolog 2 (SPNS2) transporter in H-MVEC is crucial for MAB migration measured by Boyden chambers and for the formation and stabilization of capillary-like structures in a 3D Matrigel culture. Moreover, the conditioned medium (CM) harvested from H-MVEC, where SK1, SK2, and SPNS2 were down-regulated, exerted a significantly diminished effect on MAB capillary morphogenesis and migration. Notably, we demonstrated that S1P1 and S1P3 receptors were positively involved in CM-induced capillary-like formation and migration, while S1P2 exerted a negative role on CM-induced migratory action of MAB. Finally, SK inhibition as well as MAB S1P1 and S1P3 down-regulation impaired H-MVEC-MAB cross-talk significantly reducing in vivo angiogenesis evaluated by Matrigel plug assay. These findings individuate novel targets for the employment of MAB in vascular-related pathologic conditions. • Down-regulation of SK1/2 in H-MVEC impaired vessel formation when cultured with MAB. • H-MVEC SPNS2 is critical for morphogenesis and migration induced by H-MVEC CM of MAB. • CM from SK1- and SK2-siRNA H-MVEC impaired morphogenesis and migration of MAB.

  12. Pb2+ induces gastrin gene expression by extracellular signal-regulated kinases 1/2 and transcription factor activator protein 1 in human gastric carcinoma cells.

    Science.gov (United States)

    Chan, Chien-Pin; Tsai, Yao-Ting; Chen, Yao-Li; Hsu, Yu-Wen; Tseng, Joseph T; Chuang, Hung-Yi; Shiurba, Robert; Lee, Mei-Hsien; Wang, Jaw-Yuan; Chang, Wei-Chiao

    2015-02-01

    Divalent lead ions (Pb(2+) ) are toxic environmental pollutants known to cause serious health problems in humans and animals. Absorption of Pb(2+) from air, water, and food takes place in the respiratory and digestive tracts. The ways in which absorbed Pb(2+) affects cell physiology are just beginning to be understood at the molecular level. Here, we used reverse transcription PCR and Western blotting to analyze cultures of human gastric carcinoma cells exposed to 10 μM lead nitrate. We found that Pb(2+) induces gastrin hormone gene transcription and translation in a time-dependent manner. Promoter deletion analysis revealed that activator protein 1 (AP1) was necessary for gastrin gene transcription in cells exposed to Pb(2+) . MitogIen-activated protein kinase (MAPK)/ERK kinase inhibitor PD98059 suppressed the Pb(2+) -induced increase in messenger RNA. Epidermal growth factor receptor (EGFR) inhibitors AG1478 and PD153035 reduced both transcription and phosphorylation by extracellular signal-regulated kinase (ERK1/2). Cells exposed to Pb(2+) also increased production of c-Jun protein, a component of AP1, and over-expression of c-Jun enhanced activation of the gastrin promoter. In sum, the findings suggest the EGFR-ERK1/2-AP1 pathway mediates the effects of Pb(2+) on gastrin gene activity in cell culture. © 2013 Wiley Periodicals, Inc.

  13. Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells.

    Science.gov (United States)

    Guon, Tae Eun; Chung, Ha Sook

    2017-08-01

    The present study was performed to determine the effect of Moringa oleifera fruit extract on the apoptosis of human melanoma A2058 cells. A2058 cells were treated for 72 h with Moringa oleifera fruit extract at 50-100 µg/ml, and cell viability with apoptotic changes was examined. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) was examined. It was revealed that Moringa oleifera fruit extract significantly inhibited the cell viability and promoted apoptosis of A2058 cells in a concentration-dependent manner. Moringa oleifera fruit extract-treated A2058 cells exhibited increased activities of cleaved caspase-9 and caspase-3. It also caused an enhancement of MAPK phosphorylation and ROS production. The pro-apoptotic activity of Moringa oleifera fruit extract was significantly reversed by pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125, extracellular-signal-regulated kinase (ERK) inhibitor PD98058 or ROS inhibitor N-acetyl-L-cysteine. Taken together, Moringa oleifera fruit extract is effective in inducing mitochondrial apoptosis of A2058 cells, which is mediated through induction of ROS formation, and JNK and ERK activation. Moringa oleifera fruit extract may thus have therapeutic benefits for human melanoma A2058 cells.

  14. Structure of the Human Protein Kinase CK2 Catalytic Subunit CK2α′ and Interaction Thermodynamics with the Regulatory Subunit CK2β

    DEFF Research Database (Denmark)

    Bischoff, Nils; Olsen, Birgitte Brinkmann; Raaf, Jennifer

    2011-01-01

    Protein kinase CK2 (formerly "casein kinase 2") is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement...... of CK2α' for CK2β is about 12 times lower than that of CK2α and is less driven by enthalpy. This result fits the observation that the β4/β5 loop, a key element of the CK2α/CK2β interface, adopts an open conformation in CK2α', while in CK2α, it opens only after assembly with CK2β. The open β4/β5 loop...

  15. Epigallocatechin-3-gallate blocks triethylene glycol dimethacrylate-induced cyclooxygenase-2 expression by suppressing extracellular signal-regulated kinase in human dental pulp and embryonic palatal mesenchymal cells.

    Science.gov (United States)

    Yang, Wan-Hsien; Deng, Yi-Ting; Kuo, Mark Yen-Ping; Liu, Cheing-Meei; Chang, Hao-Hueng; Chang, Jenny Zwei-Chieng

    2013-11-01

    Methacrylate resin-based materials could release components into adjacent environment even after polymerization. The major components leached include triethylene glycol dimethacrylate (TEGDMA). TEGDMA has been shown to induce the expression of cyclooxygenase-2 (COX-2). However, the mechanisms are not completely understood. The aims of this study were to investigate the molecular mechanism underlying TEGDMA-induced COX-2 in 2 oral cell types, the primary culture of human dental pulp (HDP) cells and the human embryonic palatal mesenchymal (HEPM) pre-osteoblasts, and to propose potential strategy to prevent or ameliorate the TEGDMA-induced inflammation in oral tissues. TEGDMA-induced COX-2 expression and its signaling pathways were assessed by Western blot analyses in HDP and HEPM cells. The inhibition of TEGDMA-induced COX-2 protein expression using various dietary phytochemicals was investigated. COX-2 protein expression was increased after exposure to TEGDMA at concentrations as low as 5 μmol/L. TEGDMA-induced COX-2 expression was associated with reaction oxygen species, the extracellular signal-regulated kinase 1/2, and the p38 mitogen-activated protein kinase signaling pathways in HDP and HEPM cells. The activation of p38 mitogen-activated protein kinase was directly associated with reactive oxygen species. Epigallocatechin-3-gallate suppressed TEGDMA-induced COX-2 expression by inhibiting phosphorylation of extracellular signal-regulated kinase 1/2. Cells exposed to low concentrations of TEGDMA may induce inflammatory responses of the adjacent tissues, and this should be taken into consideration during common dental practice. Green tea, which has a long history of safe beverage consumption, may be a useful agent for the prevention or treatment of TEGDMA-induced inflammation in oral tissues. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  16. Cytotoxic Activity of 3,6-Dihydroxyflavone in Human Cervical Cancer Cells and Its Therapeutic Effect on c-Jun N-Terminal Kinase Inhibition

    Directory of Open Access Journals (Sweden)

    Eunjung Lee

    2014-08-01

    Full Text Available Previously we have shown that 3,6-dihydroxyflavone (3,6-DHF is a potent agonist of the human peroxisome proliferator-activated receptor (hPPAR with cytotoxic effects on human cervical cancer cells. To date, the mechanisms by which 3,6-DHF exerts its antitumor effects on cervical cells have not been clearly defined. Here, we demonstrated that 3,6-DHF exhibits a novel antitumor activity against HeLa cells with IC50 values of 25 μM and 9.8 μM after 24 h and 48 h, respectively. We also showed that the anticancer effects of 3,6-DHF are mediated via the toll-like receptor (TLR 4/CD14, p38 mitogen-activated protein kinase (MAPK, Jun-N terminal kinase (JNK, extracellular-signaling regulated kinase (ERK, and cyclooxygenase (COX-2 pathways in lipopolysaccharide (LPS-stimulated RAW264.7 cells. We found that 3,6-DHF showed a similar IC50 (113 nM value to that of the JNK inhibitor, SP600125 (IC50 = 118 nM in a JNK1 kinase assay. Binding studies revealed that 3,6-DHF had a strong binding affinity to JNK1 (1.996 × 105 M−1 and that the 6-OH and the carbonyl oxygen of the C ring of 3,6-DHF participated in hydrogen bonding interactions with the carbonyl oxygen and the amide proton of Met111, respectively. Therefore, 3,6-DHF may be a candidate inhibitor of JNKs, with potent anticancer effects.

  17. 5-Aza-2'-deoxycytidine protects against emphysema in mice via suppressing p16Ink4a expression in lung tissue

    Directory of Open Access Journals (Sweden)

    He ZH

    2017-10-01

    Full Text Available Zhi-Hui He,1 Yan Chen,2 Ping Chen,2 Sheng-Dong He,2 Hui-Hui Zeng,2 Ji-Ru Ye,2 Da Liu,2 Jun Cao3 1Intensive Care Unit, 2Department of Respiratory Medicine, Second Xiangya Hospital, Central South University, Changsha, 3Department of Respiratory Medicine, Hunan Provincial People’s Hospital, Changsha, China Background: There is a growing realization that COPD, or at least emphysema, involves several processes presenting in aging and cellular senescence. Endothelial progenitor cells (EPCs contribute to neovascularization and play an important role in the development of COPD. The gene for p16Ink4a is a major dominant senescence one. The aim of the present study was to observe changes in lung function, histomorphology of lung tissue, and expression of p16Ink4a in lung tissue and bone marrow-derived EPCs in emphysematous mice induced by cigarette-smoke extract (CSE, and further to search for a potential candidate agent protecting against emphysema induced by CSE. Materials and methods: An animal emphysema model was induced by intraperitoneal injection of CSE. 5-Aza-2'-deoxycytidine (5-Aza-CdR was administered to the emphysematous mice. Lung function and histomorphology of lung tissue were measured. The p16Ink4a protein and mRNA in EPCs and lung tissues were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction, respectively. Results: CSE induced emphysema with increased p16Ink4a expression in lung tissue and bone marrow-derived EPCs. 5-Aza-CdR partly protected against emphysema, especially in the lung-morphology profile, and partly protest against the overexpression of p16Ink4a in EPCs and lung tissue induced by CSE. Conclusion: 5-Aza-CdR partly protected against emphysema in mice via suppressing p16Ink4a expression in EPCs and lung tissue. Keywords: 5-Aza-2'-deoxycytidine, cigarette smoke, emphysema, endothelial progenitor cells, p16Ink4a

  18. Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells.

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    Jie Hong

    Full Text Available Mechanisms of the progression from Barrett's esophagus (BE to esophageal adenocarcinoma (EA are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

  19. Deoxyribonucleoside kinases activate nucleoside antibiotics in severely pathogenic bacteria.

    Science.gov (United States)

    Sandrini, Michael P B; Shannon, Oonagh; Clausen, Anders R; Björck, Lars; Piskur, Jure

    2007-08-01

    Common bacterial pathogens are becoming progressively more resistant to traditional antibiotics, representing a major public-health crisis. Therefore, there is a need for a variety of antibiotics with alternative modes of action. In our study, several nucleoside analogs were tested against pathogenic staphylococci and streptococci. We show that pyrimidine-based nucleoside analogs, like 3'-azido-3'-deoxythymidine (AZT) and 2',2'-difluoro-2'deoxycytidine (gemcitabine), are specifically activated by the endogenous bacterial deoxyribonucleoside kinases, leading to cell death. Deoxyribonucleoside kinase-deficient Escherichia coli strains become highly susceptible to nucleoside analogs when they express recombinant kinases from Staphylococcus aureus or Streptococcus pyogenes. We further demonstrate that recombinant S. aureus deoxyadenosine kinase efficiently phosphorylates the anticancer drug gemcitabine in vitro and is therefore the key enzyme in the activation pathway. When adult mice were infected intraperitoneally with a fatal dose of S. pyogenes strain AP1 and afterwards received gemcitabine, they failed to develop a systemic infection. Nucleoside analogs may therefore represent a promising alternative for combating pathogenic bacteria.

  20. AMG 900, a potent inhibitor of aurora kinases causes pharmacodynamic changes in p-Histone H3 immunoreactivity in human tumor xenografts and proliferating mouse tissues.

    Science.gov (United States)

    Juan, Gloria; Bush, Tammy L; Ma, Connie; Manoukian, Raffi; Chung, Grace; Hawkins, Jennifer M; Zoog, Stephen; Kendall, Richard; Radinsky, Robert; Loberg, Robert; Friberg, Greg; Payton, Marc

    2014-11-04

    The Aurora family of serine-threonine kinases are essential regulators of cell division in mammalian cells. Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis. AMG 900 is a highly potent and selective pan-aurora kinase inhibitor that has entered clinical evaluation in adult patients with advanced cancers. In mice, oral administration of AMG 900 blocks the phosphorylation of histone H3 on serine-10 (p-Histone H3), a proximal substrate of aurora-B and inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models. In order to establish a preclinical pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 that could be translated to the clinic, we used flow cytometry and laser scanning cytometry detection platforms to assess the effects on p-Histone H3 inhibition in terms of sensitivity, precision, and specificity, in human tumor xenografts in conjunction with mouse skin and bone marrow tissues. Mice with established COLO 205 tumors were administered AMG 900 at 3.75, 7.5, and 15 mg/kg and assessed after 3 hours. Significant suppression of p-Histone H3 in mouse skin was only observed at 15 mg/kg (p Histone H3 in tumors and surrogate tissues (although tissues such as skin may be less sensitive for assessing PD effects). To further extend our work, we evaluated the feasibility of measuring p-Histone H3 using fine-needle aspirate (FNA) tumor xenograft biopsies. Treatment with AMG 900 significantly inhibited p-Histone H3 (>99% inhibition, p Histone H3 positive cells using mock FNAs from primary human breast tumor tissues. Phosphorylation of histone H3 is a useful biomarker to determine the pharmacodynamics (PD) activity of AMG 900. FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.

  1. Protein kinase A enhances lipopolysaccharide-induced IL-6, IL-8, and PGE2 production by human gingival fibroblasts

    Directory of Open Access Journals (Sweden)

    Ara Toshiaki

    2012-03-01

    Full Text Available Abstract Objective Periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. Interleukin (IL-6, IL-8, and the chemical mediator prostaglandin E2 (PGE2 are known to play important roles in inflammatory responses and tissue degradation. Recently, we reported that the protein kinase A (PKA inhibitor H-89 suppresses lipopolysaccharide (LPS-induced IL-8 production by human gingival fibroblasts (HGFs. In the present study, the relevance of the PKA activity and two PKA-activating drugs, aminophylline and adrenaline, to LPS-induced inflammatory cytokines (IL-6 and IL-8 and PGE2 by HGFs were examined. Methods HGFs were treated with LPS from Porphyromonas gingivalis and H-89, the cAMP analog dibutyryl cyclic AMP (dbcAMP, aminophylline, or adrenaline. After 24 h, IL-6, IL-8, and PGE2 levels were evaluated by ELISA. Results H-89 did not affect LPS-induced IL-6 production, but suppressed IL-8 and PGE2 production. In contrast, dbcAMP significantly increased LPS-induced IL-6, IL-8, and PGE2 production. Up to 10 μg/ml of aminophylline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at 100 μg/ml. Similarly, 0.01 μg/ml of adrenaline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at concentrations of 0.1 and 1 μg/ml. In the absence of LPS, H-89, dbcAMP, aminophylline, and adrenaline had no relevance to IL-6, IL-8, or PGE2 production. Conclusion These results suggest that the PKA pathway, and also PKA-activating drugs, enhance LPS-induced IL-6, IL-8, and PGE2 production by HGFs. However, aminophylline may not have an effect on the production of these molecules at concentrations used in clinical settings (8 to 20 μg/ml in serum. These results suggest that aminophylline does not affect inflammatory responses in periodontal disease.

  2. Identification of Phosphoglycerate Kinase 1 (PGK1 as a reference gene for quantitative gene expression measurements in human blood RNA

    Directory of Open Access Journals (Sweden)

    Unger Elizabeth R

    2011-09-01

    Full Text Available Abstract Background Blood is a convenient sample and increasingly used for quantitative gene expression measurements with a variety of diseases including chronic fatigue syndrome (CFS. Quantitative gene expression measurements require normalization of target genes to reference genes that are stable and independent from variables being tested in the experiment. Because there are no genes that are useful for all situations, reference gene selection is an essential step to any quantitative reverse transcription-PCR protocol. Many publications have described appropriate genes for a wide variety of tissues and experimental conditions, however, reference genes that may be suitable for the analysis of CFS, or human blood RNA derived from whole blood as well as isolated peripheral blood mononuclear cells (PBMCs, have not been described. Findings Literature review and analyses of our unpublished microarray data were used to narrow down the pool of candidate reference genes to six. We assayed whole blood RNA from Tempus tubes and cell preparation tube (CPT-collected PBMC RNA from 46 subjects, and used the geNorm and NormFinder algorithms to select the most stable reference genes. Phosphoglycerate kinase 1 (PGK1 was one of the optimal normalization genes for both whole blood and PBMC RNA, however, additional genes differed for the two sample types; Ribosomal protein large, P0 (RPLP0 for PBMC RNA and Peptidylprolyl isomerase B (PPIB for whole blood RNA. We also show that the use of a single reference gene is sufficient for normalization when the most stable candidates are used. Conclusions We have identified PGK1 as a stable reference gene for use with whole blood RNA and RNA derived from PBMC. When stable genes are selected it is possible to use a single gene for normalization rather than two or three. Optimal normalization will improve the ability of results from PBMC RNA to be compared with those from whole blood RNA and potentially allows comparison of

  3. Isoliquiritigenin induces apoptosis of human bladder cancer T24 cells via a cyclin-dependent kinase-independent mechanism.

    Science.gov (United States)

    Si, Lingling; Yang, Xinhui; Yan, Xinyan; Wang, Yanming; Zheng, Qiusheng

    2017-07-01

    The aim of the present study was to investigate whether an increase in cyclin-dependent kinase 2 (CDK2) activity is involved in apoptosis of human bladder cancer T24 cells induced by isoliquiritigenin (ISL). The viability of T24 cells was estimated using a sulforhodamine B assay. Cell morphological changes were examined using Hoechst 33258 staining. The apoptotic rate was determined by staining cells with Annexin V-fluorescein isothiocyanate and propidium iodide labeling. The mitochondrial membrane potential (ΔΨm) was measured using 5,5,6,6-tetrachloro-1,1, 3,3-tetraethyl benzimidazole carbocyanine iodide. Alterations in the apoptosis-related regulators B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Bcl-2-interacting mediator of cell death (Bim), apoptotic protease-activating facter-1 (Apaf-1), caspase-9 and caspase-3 were determined using reverse transcription-polymerase chain reaction (PCR) and quantitative PCR methods. Western blot analysis was used to detect the expression of Bcl-2, Bax and caspase-3. CDK2 activity was measured using a spectrometric assay. Following treatment with ISL (between 30 and 70 µg/ml) for 24 h, typical apoptotic morphological changes were observed in T24 cells, exhibiting an edge set of chromosomes, nuclear condensation, nuclear fragmentation and other morphological features. Treatment with ISL increased the apoptotic ratio of T24 cells in a concentration-dependent manner and induced a decrease in the ΔΨm in a time-dependent manner. Treatment with ISL upregulated the expression of Bax, Bim, Apaf-1, caspase-9 and caspase-3, downregulated the expression of Bcl-2, and increased CDK2 activity. MK-8776 (an inhibitor of CDK2) antagonized the apoptosis induced by ISL, and, compared with treatment with ISL alone, pretreatment with MK-8776 inhibited the decrease in ΔΨm, downregulated the mRNA expression of Bax, Bim, Apaf-1, caspase-9 and caspase-3, and upregulated Bcl-2 mRNA expression. Western blot analysis demonstrated

  4. Human chorionic gonadotropin stimulates spheroid attachment on fallopian tube epithelial cells through the mitogen-activated protein kinase pathway and down-regulation of olfactomedin-1.

    Science.gov (United States)

    So, Kam-Hei; Kodithuwakku, Suranga P; Kottawatta, Kottawattage S A; Li, Raymond H W; Chiu, Philip C N; Cheung, Annie N Y; Ng, Ernest H Y; Yeung, William S B; Lee, Kai-Fai

    2015-08-01

    To study the effect of human chorionic gonadotropin (hCG) on olfactomedin-1 (Olfm1) expression and spheroid attachment in human fallopian tube epithelial cells in vitro. Experimental study. Reproductive biology laboratory. Healthy nonpregnant women. No patient interventions. Luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and Olfm1 expression in fallopian tube epithelium cell line (OE-E6/E7 cells). OE-E6/E7 cells treated with hCG, U0126 extracellular signal-regulated kinase (ERK) inhibitor, or XAV939 Wnt/β-catenin inhibitor were analyzed by Western blotting, real-time polymerase chain reaction, and in vitro spheroid attachment assay. Human chorionic gonadotropin increased spheroid attachment on OE-E6/E7 cells through down-regulation of Olfm1 and activation of Wnt and mitogen-activated protein kinase (MAPK) signaling pathways. U0126 down-regulated both MAPK and Wnt/β-catenin signaling pathways and up-regulated Olfm1 expression. XAV939 down-regulated only the Wnt/β-catenin signaling pathway but up-regulated Olfm1 expression. Human chorionic gonadotropin activated both ERK and Wnt/β-catenin signaling pathways and enhanced spheroid attachment on fallopian tube epithelial cells through down-regulation of Olfm1 expression. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  5. Association of SRC-related kinase Lyn with the interleukin-2 receptor and its role in maintaining constitutive phosphorylation of JAK/STAT in human T-cell leukemia virus type 1-transformed T cells.

    Science.gov (United States)

    Shuh, Maureen; Morse, Barry A; Heidecker, Gisela; Derse, David

    2011-05-01

    Human T-cell leukemia virus type 1 (HTLV-1) infection and transformation are associated with an incremental switch in the expression of the Src-related protein tyrosine kinases Lck and Lyn. We examined the physical and functional interactions of Lyn with receptors and signal transduction proteins in HTLV-1-infected T cells. Lyn coimmunoprecipitates with the interleukin-2 beta receptor (IL-2Rβ) and JAK3 proteins; however, the association of Lyn with the IL-2Rβ and Lyn kinase activity was independent of IL-2 stimulation. Phosphorylation of Janus kinase 3 (JAK3) and signal transducers and activator of transcription 5 (STAT5) proteins was reduced by treatment of cells with the Src kinase inhibitor PP2 or by ectopic expression of a dominant negative Lyn kinase protein.

  6. HER family kinase domain mutations promote tumor progression and can predict response to treatment in human breast cancer

    KAUST Repository

    Boulbes, Delphine R.

    2014-11-11

    Resistance to HER2-targeted therapies remains a major obstacle in the treatment of HER2-overexpressing breast cancer. Understanding the molecular pathways that contribute to the development of drug resistance is needed to improve the clinical utility of novel agents, and to predict the success of targeted personalized therapy based on tumor-specific mutations. Little is known about the clinical significance of HER family mutations in breast cancer. Because mutations within HER1/EGFR are predictive of response to tyrosine kinase inhibitors (TKI) in lung cancer, we investigated whether mutations in HER family kinase domains are predictive of response to targeted therapy in HER2-overexpressing breast cancer. We sequenced the HER family kinase domains from 76 HER2-overexpressing invasive carcinomas and identified 12 missense variants. Patients whose tumors carried any of these mutations did not respond to HER2 directed therapy in the metastatic setting. We developed mutant cell lines and used structural analyses to determine whether changes in protein conformation could explain the lack of response to therapy. We also functionally studied all HER2 mutants and showed that they conferred an aggressive phenotype and altered effects of the TKI lapatinib. Our data demonstrate that mutations in the finely tuned HER kinase domains play a critical function in breast cancer progression and may serve as prognostic and predictive markers.

  7. Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration

    NARCIS (Netherlands)

    Siudeja, Katarzyna; Srinivasan, Balaji; Xu, Lanjun; Rana, Anil; de Jong, Jannie; Nollen, Ellen A. A.; Jackowski, Suzanne; Sanford, Lynn; Hayflick, Susan; Sibon, Ody C. M.

    2011-01-01

    Pantothenate kinase-associated neurodegeneration (PKAN is a neurodegenerative disease with unresolved pathophysiology. Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN. Coenzyme A is required for acetyl-Coenzyme A synthesis and acyl groups from the latter are

  8. Protein kinase CK2 inhibition is associated with the destabilization of HIF-1α in human cancer cells

    DEFF Research Database (Denmark)

    Guerra, Barbara; Rasmussen, Tine D. L.; Schnitzler, Alexander

    2015-01-01

    Screening for protein kinase CK2 inhibitors of the structural diversity compound library (DTP NCI/NIH) led to the discovery of 4-[(E)-(fluoren-9-ylidenehydrazinylidene)-methyl]benzoic acid (E9). E9 induces apoptotic cell death in various cancer cell lines and upon hypoxia, the compound suppresses...

  9. Irofulven (6-hydroxymethylacylfulvene, MGI 114)-induced apoptosis in human pancreatic cancer cells is mediated by ERK and JNK kinases.

    Science.gov (United States)

    Wang, Weixin; Waters, Stephen J; MacDonald, John R; Roth, Caleb; Shentu, Shujun; Freeman, James; Von Hoff, Daniel D; Miller, Alexander R

    2002-01-01

    Pancreatic carcinoma resists chemotherapeutic mediation of apoptosis. Irofulven (MGI 114, 6-hydroxymethylacylfulvene) is a novel illudin S analogue that we have shown to induce caspase-mediated apoptosis in pancreatic carcinoma cell lines. Westem blot analysis and kinase assays were used to demonstrate the activation of Erk 1/2 and JNK1 kinases following Irofulven administration in the presence and absence of selective kinase inhibitors. Irofulven activates JNK1 and Erk1/2, but not p38. The addition of the MAPK inhibitors, SB202190 and PD98059 (targeting JNK1 and Erk1/2 activation, respectively), prevents kinase activation and blocks Irofulven-induced activation of caspases -3, -7, -8 and -9. Blockade of either JNK1 or Erk1/2 results in a 50% decrease in apoptosis in MiaPaCa-2 cells treated with Irofulven. Our data demonstrated that JNK1 and Erk1/2 are activated by Irofulven treatment and that blockade of either MAPK subfamily decreases apoptosis by rendering Irofulven incapable of inducing caspase activation.

  10. Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases.

    NARCIS (Netherlands)

    Ritzerfeld, J.; Remmele, S.; Wang, T.; Temmerman, K.; Brugger, B.; Wegehingel, S.; Tournaviti, S.; Strating, J.R.P.M.; Wieland, F.T.; Neumann, B.; Ellenberg, J.; Lawerenz, C.; Hesser, J.; Erfle, H.; Pepperkok, R.; Nickel, W.

    2011-01-01

    SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary

  11. wKinMut: An integrated tool for the analysis and interpretation of mutations in human protein kinases

    DEFF Research Database (Denmark)

    Gonzalez-Izarzugaza, Jose Maria; Vazquez, Miguel; del Pozo, Angela

    2013-01-01

    Background Protein kinases are involved in relevant physiological functions and a broad number of mutations in this superfamily have been reported in the literature to affect protein function and stability. Unfortunately, the exploration of the consequences on the phenotypes of each individual...

  12. Epigenetic therapy of non-small cell lung cancer using decitabine (5-aza-2’-deoxycytidine, DAC

    Directory of Open Access Journals (Sweden)

    Richard L Momparler

    2013-07-01

    Full Text Available Epigenetic analysis shows that many genes that suppress malignancy are silenced by aberrant DNA methylation in lung cancer. Many of these genes are interesting targets for reactivation by the inhibitor of DNA methylation, decitabine (5-aza-2’-deoxycytidine, DAC. A pilot study on intense dose DAC showed promising results in patients with metastatic non-small cell lung cancer (NSCLC. However, subsequent clinical studies using low dose DAC were not very effective against NSCLC and interest in this therapy diminished. Recently, interesting responses were observed in a patient with NSCLC following treatment with a combination of the related inhibitor of DNA methylation, 5-azacytidine and an inhibitor of histone deacetylation. This finding has generated a renewed interest in the epigenetic therapy of lung cancer. Preclinical studies indicate that DAC has remarkable chemotherapeutic potential for tumor therapy. This epigenetic agent has a delayed and prolonged epigenetic action on tumor cells. This delayed action should be taken into consideration in the design and evaluation of clinical studies on DAC. Future research should be directed at finding the optimal dose-schedule of de DAC for the treatment of NSCLC.

  13. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    Science.gov (United States)

    Olesen, Sanne H.; Ingles, Donna J.; Zhu, Jin-Yi; Martin, Mathew P.; Betzi, Stephane; Georg, Gunda I.; Tash, Joseph S.; Schönbrunn, Ernst

    2015-01-01

    The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in-vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands. PMID:25608045

  14. Gastrin decreases Na+,K+-ATPase activity via a PI 3-kinase- and PKC-dependent pathway in human renal proximal tubule cells.

    Science.gov (United States)

    Liu, Tianbing; Konkalmatt, Prasad R; Yang, Yu; Jose, Pedro A

    2016-04-01

    The natriuretic effect of gastrin suggests a role in the coordinated regulation of sodium balance by the gastrointestinal tract and the kidney. The renal molecular targets and signal transduction pathways for such an effect of gastrin are largely unknown. Recently, we reported that gastrin induces NHE3 phosphorylation and internalization via phosphatidylinositol (PI) 3-kinase and PKCα. In this study, we show that gastrin induced the phosphorylation of human Na(+),K(+)-ATPase at serine 16, resulting in its endocytosis via Rab5 and Rab7 endosomes. The gastrin-stimulated phosphorylation of Na(+),K(+)-ATPase was dependent on PI 3-kinase because the phosphorylation was blocked by the PI 3-kinase inhibitor wortmannin. The phosphorylation of Na(+),K(+)-ATPase was also blocked by chelerythrine, a pan-PKC inhibitor, Gö-6976, a conventional PKC (cPKC) inhibitor, and BAPTA-AM, an intracellular calcium chelator, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The gastrin-mediated phosphorylation of Na(+),K(+)-ATPase was also inhibited by U-73122, a phospholipase C (PLC) inhibitor. These results suggest that gastrin regulates sodium hydrogen exchanger and pump in renal proximal tubule cells at the apical and basolateral membranes. Copyright © 2016 the American Physiological Society.

  15. Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2.

    Science.gov (United States)

    Olesen, Sanne H; Ingles, Donna J; Zhu, Jin-Yi; Martin, Mathew P; Betzi, Stephane; Georg, Gunda I; Tash, Joseph S; Schönbrunn, Ernst

    2015-01-19

    The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

  16. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    Directory of Open Access Journals (Sweden)

    Sanne H. Olesen

    2015-01-01

    Full Text Available The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2 did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM, while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

  17. Effect of Saturated Stearic Acid on MAP Kinase and ER Stress Signaling Pathways during Apoptosis Induction in Human Pancreatic β-Cells Is Inhibited by Unsaturated Oleic Acid

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    Jan Šrámek

    2017-11-01

    Full Text Available It has been shown that saturated fatty acids (FAs have a detrimental effect on pancreatic β-cells function and survival, leading to apoptosis, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction and regulation by FAs in β-cells remain unclear; however, mitogen-activated protein (MAP kinase and endoplasmic reticulum (ER stress signaling pathways may be involved. In this study, we tested how unsaturated oleic acid (OA affects the effect of saturated stearic acid (SA on the p38 mitogen-activated protein kinase (MAPK and extracellular signal-regulated kinase (ERK pathways as well as the ER stress signaling pathways during apoptosis induction in the human pancreatic β-cells NES2Y. We demonstrated that OA is able to inhibit all effects of SA. OA alone has only minimal or no effects on tested signaling in NES2Y cells. The point of OA inhibitory intervention in SA-induced apoptotic signaling thus seems to be located upstream of the discussed signaling pathways.

  18. Barium Promotes Anchorage-Independent Growth and Invasion of Human HaCaT Keratinocytes via Activation of c-SRC Kinase

    Science.gov (United States)

    Thang, Nguyen Dinh; Yajima, Ichiro; Kumasaka, Mayuko Y.; Ohnuma, Shoko; Yanagishita, Takeshi; Hayashi, Rumiko; Shekhar, Hossain U.; Watanabe, Daisuke; Kato, Masashi

    2011-01-01

    Explosive increases in skin cancers have been reported in more than 36 million patients with arsenicosis caused by drinking arsenic-polluted well water. This study and previous studies showed high levels of barium as well as arsenic in the well water. However, there have been no reports showing a correlation between barium and cancer. In this study, we examined whether barium (BaCl2) may independently have cancer-related effects on human precancerous keratinocytes (HaCaT). Barium (5–50 µM) biologically promoted anchorage-independent growth and invasion of HaCaT cells in vitro. Barium (5 µM) biochemically enhanced activities of c-SRC, FAK, ERK and MT1-MMP molecules, which regulate anchorage-independent growth and/or invasion. A SRC kinase specific inhibitor, protein phosphatase 2 (PP2), blocked barium-mediated promotion of anchorage-independent growth and invasion with decreased c-SRC kinase activity. Barium (2.5–5 µM) also promoted anchorage-independent growth and invasion of fibroblasts (NIH3T3) and immortalized nontumorigenic melanocytes (melan-a), but not transformed cutaneous squamous cell carcinoma (HSC5 and A431) and malignant melanoma (Mel-ret) cells, with activation of c-SRC kinase. Taken together, our biological and biochemical findings newly suggest that the levels of barium shown in drinking well water independently has the cancer-promoting effects on precancerous keratinocytes, fibroblast and melanocytes in vitro. PMID:22022425

  19. 5-Azacytidine regulates matrix metalloproteinase-9 expression, and the migration and invasion of human fibrosarcoma HT1080 cells via PI3-kinase and ERK1/2 pathways.

    Science.gov (United States)

    Yu, Seon-Mi; Kim, Song Ja

    2016-09-01

    Abnormal methylation of promoter CpG islands is one of the hallmarks of cancer cells, and is catalyzed by DNA methyltransferases. 5-azacytidine (5-aza C), a methyltransferase inhibitor, can cause demethylation of promoter regions of diverse genes. Epigenetic processes contribute to the regulation of matrix metalloproteinase (MMP) expression. However, little is known about the mechanisms and effects of 5-aza C on the invasive and migratory capacities of human fibrosarcoma HT1080 cells. In the present study, we found that 5-aza C induces MMP-9 activity, as determined by zymography. HT1080 cell proliferation was determined following 5-aza C administration by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle was examined by flow cytometry. 5-aza C treatment inhibited cell proliferation without affecting cell viability. Furthermore, 5-aza C significantly promoted migration and invasion of HT1080 cells. 5-aza C treatment enhanced phosphorylation of extracellular signal-regulated kinase (ERK) and phosphoinositide (PI)3-kinase/Akt, and their inhibitors blocked MMP-9 activity induction, and cellular invasion and migration. Together, these findings suggest that promoter methylation may be one of the mechanisms modulating MMP-9 levels in HT1080 cells, and that 5-aza C-induced MMP-9 production is associated with the activation of ERK and PI3-kinase/Akt signaling pathways.

  20. Pharmacokinetic and pharmacodynamic analysis of 5-aza-2’-deoxycytidine (decitabine in the design of its dose-schedule for cancer therapy

    Directory of Open Access Journals (Sweden)

    Karahoca Metin

    2013-02-01

    Full Text Available Abstract 5-Aza-2′-deoxycytidine (5-AZA-CdR, decitabine, an epigenetic drug that inhibits DNA methylation, is currently used to treat myelodysplastic syndrome (MDS, and is under investigation for treating acute myeloid leukemia (AML and other malignancies. 5-AZA-CdR can reactivate tumor suppressor genes silenced by aberrant DNA methylation, a frequent event in all types of cancer. Because this epigenetic change is reversible, it is a good target for 5-AZA-CdR therapy. We have reviewed the preclinical data of 5-AZA-CdR to analyze the concentrations and exposure times required to eradicate cancer stem cells. We analyzed the dose-schedules used in animal models that show potent antineoplastic activity of 5-AZA-CdR. We attempted to correlate the preclinical data with the responses obtained in clinical trials of 5-AZA-CdR in patients with cancer. The pharmacokinetics and drug distribution of 5-AZA-CdR are key parameters because adequate therapeutic drug levels are required to eliminate cancer stem cells in all anatomic compartments. The plasma half-life of 5-AZA-CdR in humans is approximately 20 minutes due to the high levels in the liver of cytidine deaminase, the enzyme that inactivates this analogue. This provides a rationale to use an inhibitor of cytidine deaminase in combination with 5-AZA-CdR. Low-dose 5-AZA-CdR is effective for MDS and AML and can induce complete remissions (CR. However, maintenance of CR with low-dose 5-AZA-CdR is difficult. Based on analyses of preclinical and clinical data, low dose 5-AZA-CdR has the potential to be an effective form of therapy in some patients with cancer. For patients who do not respond to low dose therapy we recommend dose-intensive treatment with 5-AZA-CdR. Patients who are candidates for intensive dose 5-AZA-CdR should have a good bone marrow status so as to permit adequate recovery from myelosuppression, the major toxicity of 5-AZA-CdR. Solid tumors are also interesting targets for therapy with 5

  1. Humanized thymidine kinase-NOG mice can be used to identify drugs that cause animal-specific hepatotoxicity: a case study with furosemide.

    Science.gov (United States)

    Xu, Dan; Michie, Sara A; Zheng, Ming; Takeda, Saori; Wu, Manhong; Peltz, Gary

    2015-07-01

    Interspecies differences have limited the predictive utility of toxicology studies performed using animal species. A drug that could be a safe and effective treatment in humans could cause toxicity in animals, preventing it from being used in humans. We investigated whether the use of thymidine kinase (TK)-NOG mice with humanized livers could prevent this unfortunate outcome (i.e., "rescue" a drug for use in humans). A high dose of furosemide is known to cause severe liver toxicity in mice, but it is a safe and effective treatment in humans. We demonstrate that administration of a high dose of furosemide (200 mg/kg i.p.) causes extensive hepatotoxicity in control mice but not in humanized TK-NOG mice. This interspecies difference results from a higher rate of production of the toxicity-causing metabolite by mouse liver. Comparison of their survival curves indicated that the humanized mice were more resistant than control mice to the hepatotoxicity caused by high doses of furosemide. In this test case, humanized TK-NOG mouse studies indicate that humans could be safely treated with a high dose of furosemide. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  2. Reduced corporal fibrosis to protect erectile function by inhibiting the Rho-kinase/LIM-kinase/cofilin pathway in the aged transgenic rat harboring human tissue kallikrein 1

    Directory of Open Access Journals (Sweden)

    Kai Cui

    2017-01-01

    Full Text Available Our previous studies have demonstrated that erectile function was preserved in aged transgenic rats (TGR harboring the human tissue kallikrein 1 (hKLK1, while the molecular level of hKLK1 on corporal fibrosis to inhibit age-related erectile dysfunction (ED is poorly understood. Male wild-type Sprague-Dawley rats (WTR and TGR harboring the hKLK1 gene were fed to 4- or 18-month-old and divided into three groups: young WTR (yWTR as the control, aged WTR (aWTR, and aged TGR (aTGR. Erectile function of all rats was assessed by cavernous nerve electrostimulation method. Masson′s trichrome staining was used to evaluate corporal fibrosis in the corpus cavernosum. We found that the erectile function of rats in the aWTR group was significantly lower than that of other two groups. Masson′s trichrome staining revealed that compared with those of the yWTR and aTGR groups, the ratio of smooth muscle cell (SMC/collagen (C was significantly lower in the aWTR group. Immunohistochemistry and Western blotting analysis were performed, and results demonstrated that expression of α-SMA was lower, while expressions of transforming growth factor-β 1 (TGF-β1, RhoA, ROCK1, p-MYPT1, p-LIMK2, and p-cofilin were higher in the aWTR group compared with those in other two groups. However, LIMK2 and cofilin expressions did not differ among three groups. Taken together, these results indicated that the RhoA/ROCK1/LIMK/cofilin pathway may be involved in the corporal fibrosis caused by advanced age, and hKLK1 may reduce this corporal fibrosis by inhibiting the activation of this pathway to ameliorate age-related ED.

  3. PI 3 kinase related kinases-independent proteolysis of BRCA1 regulates Rad51 recruitment during genotoxic stress in human cells.

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    Ian Hammond-Martel

    2010-11-01

    Full Text Available The function of BRCA1 in response to ionizing radiation, which directly generates DNA double strand breaks, has been extensively characterized. However previous investigations have produced conflicting data on mutagens that initially induce other classes of DNA adducts. Because of the fundamental and clinical importance of understanding BRCA1 function, we sought to rigorously evaluate the role of this tumor suppressor in response to diverse forms of genotoxic stress.We investigated BRCA1 stability and localization in various human cells treated with model mutagens that trigger different DNA damage signaling pathways. We established that, unlike ionizing radiation, either UVC or methylmethanesulfonate (MMS (generating bulky DNA adducts or alkylated bases respectively induces a transient downregulation of BRCA1 protein which is neither prevented nor enhanced by inhibition of PIKKs. Moreover, we found that the proteasome mediates early degradation of BRCA1, BARD1, BACH1, and Rad52 implying that critical components of the homologous recombination machinery need to be functionally abrogated as part of the early response to UV or MMS. Significantly, we found that inhibition of BRCA1/BARD1 downregulation is accompanied by the unscheduled recruitment of both proteins to chromatin along with Rad51. Consistently, treatment of cells with MMS engendered complete disassembly of Rad51 from pre-formed ionizing radiation-induced foci. Following the initial phase of BRCA1/BARD1 downregulation, we found that the recovery of these proteins in foci coincides with the formation of RPA and Rad51 foci. This indicates that homologous recombination is reactivated at later stage of the cellular response to MMS, most likely to repair DSBs generated by replication blocks.Taken together our results demonstrate that (i the stabilities of BRCA1/BARD1 complexes are regulated in a mutagen-specific manner, and (ii indicate the existence of mechanisms that may be required to

  4. Reduced corporal fibrosis to protect erectile function by inhibiting the Rho-kinase/LIM-kinase/cofilin pathway in the aged transgenic rat harboring human tissue kallikrein 1

    Science.gov (United States)

    Cui, Kai; Luan, Yang; Wang, Tao; Zhuan, Li; Rao, Ke; Wang, Shao-Gang; Ye, Zhang-Qun; Liu, Ji-Hong; Wang, Dao-Wen

    2017-01-01

    Our previous studies have demonstrated that erectile function was preserved in aged transgenic rats (TGR) harboring the human tissue kallikrein 1 (hKLK1), while the molecular level of hKLK1 on corporal fibrosis to inhibit age-related erectile dysfunction (ED) is poorly understood. Male wild-type Sprague-Dawley rats (WTR) and TGR harboring the hKLK1 gene were fed to 4- or 18-month-old and divided into three groups: young WTR (yWTR) as the control, aged WTR (aWTR), and aged TGR (aTGR). Erectile function of all rats was assessed by cavernous nerve electrostimulation method. Masson's trichrome staining was used to evaluate corporal fibrosis in the corpus cavernosum. We found that the erectile function of rats in the aWTR group was significantly lower than that of other two groups. Masson's trichrome staining revealed that compared with those of the yWTR and aTGR groups, the ratio of smooth muscle cell (SMC)/collagen (C) was significantly lower in the aWTR group. Immunohistochemistry and Western blotting analysis were performed, and results demonstrated that expression of α-SMA was lower, while expressions of transforming growth factor-β 1 (TGF-β1), RhoA, ROCK1, p-MYPT1, p-LIMK2, and p-cofilin were higher in the aWTR group compared with those in other two groups. However, LIMK2 and cofilin expressions did not differ among three groups. Taken together, these results indicated that the RhoA/ROCK1/LIMK/cofilin pathway may be involved in the corporal fibrosis caused by advanced age, and hKLK1 may reduce this corporal fibrosis by inhibiting the activation of this pathway to ameliorate age-related ED. PMID:27678468

  5. The role of mitogen-activated protein kinases in crystalline silica-induced cyclooxygenase-2 expression in A549 human lung epithelial cells.

    Science.gov (United States)

    Tomaru, Makoto; Matsuoka, Masato

    2011-09-01

    We examined the role of mitogen-activated protein kinase (MAPK) signaling pathways in crystalline silica-induced expression of cyclooxygenase (COX)-2, an important mediator of airway inflammation, in A549 human lung epithelial cells. The levels of COX-2 mRNA increased after a 30-min exposure, and COX-2 protein increased after a 2-h exposure to crystalline silica. Both remained elevated at 8 h; however, no change was observed in the expression of the constitutive COX-1 isoform. The level of prostaglandin E(2), a major product of COX enzymes, increased in response to crystalline silica exposure. Phosphorylated forms of MAPKs including extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 were also increased after crystalline silica exposure. COX-2 expression was markedly suppressed by treatment with the p38 inhibitor, SB203580, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. Treatment with the nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, markedly suppressed silica-induced COX-2 expression. These results show that crystalline silica exposure induces COX-2 expression in A549 cells in a manner that is dependent on the MAPK and NF-κB pathways. Although a marked induction of MAPK phosphatase (MKP)-1 expression was observed in A549 cells exposed to crystalline silica, the silencing of MKP-1 expression using short interference RNA did not affect silica-induced COX-2 expression, suggesting that the down-regulation of COX-2 expression by MKP-1 is unlikely.

  6. Phosphatase and tensin homologue/protein kinase B pathway linked to motor neuron survival in human superoxide dismutase 1-related amyotrophic lateral sclerosis.

    Science.gov (United States)

    Kirby, Janine; Ning, Ke; Ferraiuolo, Laura; Heath, Paul R; Ismail, Azza; Kuo, Su-Wei; Valori, Chiara F; Cox, Laura; Sharrack, Basil; Wharton, Stephen B; Ince, Paul G; Shaw, Pamela J; Azzouz, Mimoun

    2011-02-01

    Gene expression profiling has been used previously with spinal cord homogenates and laser capture microdissected motor neurons to determine the mechanisms involved in neurodegeneration in amyotrophic lateral sclerosis. However, while cellular and animal model work has focused on superoxide dismutase 1-related amyotrophic lateral sclerosis, the transcriptional profile of human mutant superoxide dismutase 1 motor neurons has remained undiscovered. The aim of this study was to apply gene expression profiling to laser captured motor neurons from human superoxide dismutase 1-related amyotrophic lateral sclerosis and neurologically normal control cases, in order to determine those pathways dysregulated in human superoxide dismutase 1-related neurodegeneration and to establish potential pathways suitable for therapeutic intervention. Identified targets were then validated in cultured cell models using lentiviral vectors to manipulate the expression of key genes. Microarray analysis identified 1170 differentially expressed genes in spinal cord motor neurons from superoxide dismutase 1-related amyotrophic lateral sclerosis, compared with controls. These genes encoded for proteins in multiple functional categories, including those involved in cell survival and cell death. Further analysis determined that multiple genes involved in the phosphatidylinositol-3 kinase signalling cascade were differentially expressed in motor neurons that survived the disease process. Functional experiments in cultured cells and primary motor neurons demonstrate that manipulating this pathway by reducing the expression of a single upstream target, the negative phosphatidylinositol-3 kinase regulator phosphatase and tensin homology, promotes a marked pro-survival effect. Therefore, these data indicate that proteins in the phosphatidylinositol-3 kinase pathway could represent a target for therapeutic manipulation in motor neuron degeneration.

  7. Requirement of ERα and basal activities of EGFR and Src kinase in Cd-induced activation of MAPK/ERK pathway in human breast cancer MCF-7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Song, Xiulong, E-mail: songxiulong@hotmail.com; Wei, Zhengxi; Shaikh, Zahir A., E-mail: zshaikh@uri.edu

    2015-08-15

    Cadmium (Cd) is a common environmental toxicant and an established carcinogen. Epidemiological studies implicate Cd with human breast cancer. Low micromolar concentrations of Cd promote proliferation of human breast cancer cells in vitro. The growth promotion of breast cancer cells is associated with the activation of MAPK/ERK pathway. This study explores the mechanism of Cd-induced activation of MAPK/ERK pathway. Specifically, the role of cell surface receptors ERα, EGFR, and Src kinase was evaluated in human breast cancer MCF-7 cells treated with 1–3 μM Cd. The activation of ERK was studied using a serum response element (SRE) luciferase reporter assay. Receptor phosphorylation was detected by Western blot analyses. Cd treatment increased both the SRE reporter activity and ERK1/2 phosphorylation in a concentration-dependent manner. Cd treatment had no effect on reactive oxygen species (ROS) generation. Also, blocking the entry of Cd into the cells with manganese did not diminish Cd-induced activation of MAPK/ERK. These results suggest that the effect of Cd was likely not caused by intracellular ROS generation, but through interaction with the membrane receptors. While Cd did not appear to activate either EGFR or Src kinase, their inhibition completely blocked the Cd-induced activation of ERK as well as cell proliferation. Similarly, silencing ERα with siRNA or use of ERα antagonist blocked the effects of Cd. Based on these results, it is concluded that not only ERα, but also basal activities of EGFR and Src kinase are essential for Cd-induced signal transduction and activation of MAPK/ERK pathway for breast cancer cell proliferation. - Highlights: • Low micromolar concentrations of Cd rapidly activate ERK1/2 in MCF-7 cells. • Signal transduction and resulting cell proliferation require EGFR, ERα, and Src. • These findings implicate Cd in promotion of breast cancer.

  8. Novel covalent modification of human anaplastic lymphoma kinase (ALK and potentiation of crizotinib-mediated inhibition of ALK activity by BNP7787

    Directory of Open Access Journals (Sweden)

    Parker AR

    2015-02-01

    Full Text Available Aulma R Parker,1 Pavankumar N Petluru,1 Vicki L Nienaber,2 Min Zhao,1 Philippe Y Ayala,1 John Badger,2 Barbara Chie-Leon,2 Vandana Sridhar,2 Cheyenne Logan,2 Harry Kochat,1 Frederick H Hausheer1 1BioNumerik Pharmaceuticals, Inc., San Antonio, TX, USA; 2Zenobia Therapeutics, Inc., La Jolla, CA, USA Abstract: BNP7787 (Tavocept, disodium 2,2’-dithio-bis-ethanesulfonate is a novel, investigational, water-soluble disulfide that is well-tolerated and nontoxic. In separate randomized multicenter Phase II and Phase III clinical trials in non-small-cell lung cancer (NSCLC patients, treatment with BNP7787 in combination with standard chemotherapy resulted in substantial increases in the overall survival of patients with advanced adenocarcinoma of the lung in the first-line treatment setting. We hypothesized that BNP7787 might interact with and modify human anaplastic lymphoma kinase (ALK. At least seven different variants of ALK fusions with the gene encoding the echinoderm microtubule-associated protein-like 4 (EML4 are known to occur in NSCLC. EML4–ALK fusions are thought to account for approximately 3% of NSCLC cases. Herein, we report the covalent modification of the kinase domain of human ALK by a BNP7787-derived mesna moiety and the functional consequences of this modification in ALK assays evaluating kinase activity. The kinase domain of the ALK protein crystallizes as a monomer, and BNP7787-derived mesna-cysteine adducts were observed at Cys 1235 and Cys 1156. The BNP7787-derived mesna adduct at Cys 1156 is located in close proximity to the active site and results in substantial disorder of the P-loop and activation loop (A-loop. Comparison with the P-loop of apo-ALK suggests that the BNP7787-derived mesna adduct at Cys 1156 interferes with the positioning of Phe 1127 into a small pocket now occupied by mesna, resulting in a destabilization of the loop's binding orientation. Additionally, in vitro kinase activity assays indicate that BNP7787

  9. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells.

    Science.gov (United States)

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Zhou, Zhi-Wei; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Pan, Si-Yuan; Duan, Wei; He, Shu-Ming; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition

  10. Discovery of a novel mode of protein kinase inhibition characterized by the mechanism of inhibition of human mesenchymal-epithelial transition factor (c-Met) protein autophosphorylation by ARQ 197.

    Science.gov (United States)

    Eathiraj, Sudharshan; Palma, Rocio; Volckova, Erika; Hirschi, Marscha; France, Dennis S; Ashwell, Mark A; Chan, Thomas C K

    2011-06-10

    A number of human malignancies exhibit sustained stimulation, mutation, or gene amplification of the receptor tyrosine kinase human mesenchymal-epithelial transition factor (c-Met). ARQ 197 is a clinically advanced, selective, orally bioavailable, and well tolerated c-Met inhibitor, currently in Phase 3 clinical testing in non-small cell lung cancer patients. Herein, we describe the molecular and structural basis by which ARQ 197 selectively targets c-Met. Through our analysis we reveal a previously undisclosed, novel inhibitory mechanism that utilizes distinct regulatory elements of the c-Met kinase. The structure of ARQ 197 in complex with the c-Met kinase domain shows that the inhibitor binds a conformation that is distinct from published kinase structures. ARQ 197 inhibits c-Met autophosphorylation and is highly selective for the inactive or unphosphorylated form of c-Met. Through our analysis of the interplay between the regulatory and catalytic residues of c-Met, and by comparison between the autoinhibited canonical conformation of c-Met bound by ARQ 197 to previously described kinase domains of type III receptor tyrosine kinases, we believe this to be the basis of a powerful new in silico approach for the design of similar inhibitors for other protein kinases of therapeutic interest.

  11. Growth inhibitory effects of the dual ErbB1/ErbB2 tyrosine kinase inhibitor PKI-166 on human prostate cancer xenografts.

    Science.gov (United States)

    Mellinghoff, Ingo K; Tran, Chris; Sawyers, Charles L

    2002-09-15

    Experiments with human prostate cancer cell lines have shown that forced overexpression of the ErbB2-receptor tyrosine kinase (RTK) promotes androgen-independent growth and increases androgen receptor-transcriptional activity in a ligand-independent fashion. To investigate the relationship between ErbB-RTK signaling and androgen in genetically unmanipulated human prostate cancer, we performed biochemical and biological studies with the dual ErbB1/ErbB2 RTK inhibitor PKI-166 using human prostate cancer xenograft models with isogenic sublines reflecting the transition from androgen-dependent to androgen-independent growth. In the presence of low androgen concentrations, PKI-166 showed profound growth-inhibitory effects on tumor growth, which could be partially reversed by androgen add-back. At physiological androgen concentrations, androgen withdrawal greatly enhanced the ability of PKI-166 to retard tumor growth. The level of extracellular signal-regulated kinase activation correlated with the response to PKI-166 treatment, whereas the expression levels of ErbB1 and ErbB2 did not. These results suggest that ErbB1/ErbB2 RTKs play an important role in the biology of androgen-independent prostate cancer and provide a rationale for clinical evaluation of inhibitors targeted to this pathway.

  12. Ursolic acid-induced AMP-activated protein kinase (AMPK) activation contributes to growth inhibition and apoptosis in human bladder cancer T24 cells.

    Science.gov (United States)

    Zheng, Qing-you; Jin, Feng-suo; Yao, Chen; Zhang, Tong; Zhang, Guo-hui; Ai, Xing

    2012-03-23

    Ursolic acid (UA) has shown the anti-tumor properties against a number of human cancers both in vivo and in vitro, however, its effect in bladder cancer and the corresponding mechanisms of action remain largely unknown. Here we found that UA dose-dependently induced growth inhibition and apoptosis in human bladder cancer T24 cells, and activation of AMP-activated protein kinase (AMPK) may contribute to the process. Our Western-blot results demonstrated a significant AMPK activation after UA treatment in T24 cells. Notably, knockdown of AMPKα by the targeted shRNA largely inhibited UA-induced T24 cell growth inhibition and apoptosis, while an AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) or a constitutively active form of AMPK mimic UA's effect. We found the ceramide level was increased after UA treatment in T24 cells, and UA-induced AMPK activation and T24 cell apoptosis were inhibited by ceramide synthase inhibitor fumonisin B1, and was enhanced by exogenously adding cell permeable short-chain ceramide (C6), suggesting that ceramide might serve as an upstream signal for AMPK activation. Further, activation of AMPK by UA promoted c-Jun N-terminal kinase (JNK) activation, but inhibited mTOR complex 1 (mTORC1) signaling to cause survivin down-regulation. Our study suggests that activation of AMPK by UA contributes to growth inhibition and apoptosis in human bladder cancer cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Osthole Suppresses the Migratory Ability of Human Glioblastoma Multiforme Cells via Inhibition of Focal Adhesion Kinase-Mediated Matrix Metalloproteinase-13 Expression

    Directory of Open Access Journals (Sweden)

    Cheng-Fang Tsai

    2014-03-01

    Full Text Available Glioblastoma multiforme (GBM is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells.

  14. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    The present review on casein kinases focuses mainly on the possible metabolic role of CK-2, with special emphasis on its behavior in pathological tissues. From these data at least three ways to regulate CK-2 activity emerge: (i) CK-2 activity changes during embryogenesis, being high at certain...

  15. The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Klopffleisch, Karsten; Issinger, Olaf-Georg

    2008-01-01

    The Ser/Thr kinase CK2 (former name: casein kinase 2) is a heterotetrameric enzyme composed of two catalytic chains (CK2alpha) attached to a dimer of noncatalytic subunits. Together with the cyclin-dependent kinases and the mitogen-activated protein kinases, CK2alpha belongs to the CMGC family of...

  16. Involvement of p38 mitogen-activated protein kinase in acquired gemcitabine-resistant human urothelial carcinoma sublines

    Directory of Open Access Journals (Sweden)

    Yu-Ting Kao

    2014-07-01

    Full Text Available Resistance to chemotherapeutic drugs is one of the major challenges in the treatment of cancer. A better understanding of how resistance arises and what molecular alterations correlate with resistance is the key to developing novel effective therapeutic strategies. To investigate the underlying mechanisms of gemcitabine (Gem resistance and provide possible therapeutic options, three Gem-resistant urothelial carcinoma sublines were established (NG0.6, NG0.8, and NG1.0. These cells were cross-resistant to arabinofuranosyl cytidine and cisplatin, but sensitive to 5-fluorouracil. The resistant cells expressed lower values of [hENT1 × dCK/RRM1 × RRM2] mRNA ratio. Two adenosine triphosphate-binding cassette proteins ABCD1 as well as multidrug resistance protein 1 were elevated. Moreover, cyclin D1, cyclin-dependent kinases 2 and 4 were upregulated, whereas extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase (MAPK activity were repressed significantly. Administration of p38 MAPK inhibitor significantly reduced the Gem sensitivity in NTUB1 cells, whereas that of an extracellular signal-regulated kinase MAPK inhibitor did not. Furthermore, the Gem-resistant sublines also exhibited higher migration ability. Forced expression of p38 MAPK impaired the cell migration activity and augmented Gem sensitivity in NG1.0 cells. Taken together, these results demonstrate that complex mechanisms were merged in acquiring Gem resistance and provide information that can be important for developing therapeutic targets for treating Gem-resistant tumors.

  17. Local anesthetics induce apoptosis in human thyroid cancer cells through the mitogen-activated protein kinase pathway.

    Directory of Open Access Journals (Sweden)

    Yuan-Ching Chang

    Full Text Available Local anesthetics are frequently used in fine-needle aspiration of thyroid lesions and locoregional control of persistent or recurrent thyroid cancer. Recent evidence suggests that local anesthetics have a broad spectrum of effects including inhibition of cell proliferation and induction of apoptosis in neuronal and other types of cells. In this study, we demonstrated that treatment with lidocaine and bupivacaine resulted in decreased cell viability and colony formation of both 8505C and K1 cells in a dose-dependent manner. Lidocaine and bupivacaine induced apoptosis, and necrosis in high concentrations, as determined by flow cytometry. Lidocaine and bupivacaine caused disruption of mitochondrial membrane potential and release of cytochrome c, accompanied by activation of caspase 3 and 7, PARP cleavage, and induction of a higher ratio of Bax/Bcl-2. Based on microarray and pathway analysis, apoptosis is the prominent transcriptional change common to lidocaine and bupivacaine treatment. Furthermore, lidocaine and bupivacaine attenuated extracellular signal-regulated kinase 1/2 (ERK1/2 activity and induced activation of p38 mitogen-activated protein kinase (MAPK and c-jun N-terminal kinase. Pharmacological inhibitors of MAPK/ERK kinase and p38 MAPK suppressed caspase 3 activation and PARP cleavage. Taken together, our results for the first time demonstrate the cytotoxic effects of local anesthetics on thyroid cancer cells and implicate the MAPK pathways as an important mechanism. Our findings have potential clinical relevance in that the use of local anesthetics may confer previously unrecognized benefits in the management of patients with thyroid cancer.

  18. Local anesthetics induce apoptosis in human thyroid cancer cells through the mitogen-activated protein kinase pathway.

    Science.gov (United States)

    Chang, Yuan-Ching; Hsu, Yi-Chiung; Liu, Chien-Liang; Huang, Shih-Yuan; Hu, Meng-Chun; Cheng, Shih-Ping

    2014-01-01

    Local anesthetics are frequently used in fine-needle aspiration of thyroid lesions and locoregional control of persistent or recurrent thyroid cancer. Recent evidence suggests that local anesthetics have a broad spectrum of effects including inhibition of cell proliferation and induction of apoptosis in neuronal and other types of cells. In this study, we demonstrated that treatment with lidocaine and bupivacaine resulted in decreased cell viability and colony formation of both 8505C and K1 cells in a dose-dependent manner. Lidocaine and bupivacaine induced apoptosis, and necrosis in high concentrations, as determined by flow cytometry. Lidocaine and bupivacaine caused disruption of mitochondrial membrane potential and release of cytochrome c, accompanied by activation of caspase 3 and 7, PARP cleavage, and induction of a higher ratio of Bax/Bcl-2. Based on microarray and pathway analysis, apoptosis is the prominent transcriptional change common to lidocaine and bupivacaine treatment. Furthermore, lidocaine and bupivacaine attenuated extracellular signal-regulated kinase 1/2 (ERK1/2) activity and induced activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase. Pharmacological inhibitors of MAPK/ERK kinase and p38 MAPK suppressed caspase 3 activation and PARP cleavage. Taken together, our results for the first time demonstrate the cytotoxic effects of local anesthetics on thyroid cancer cells and implicate the MAPK pathways as an important mechanism. Our findings have potential clinical relevance in that the use of local anesthetics may confer previously unrecognized benefits in the management of patients with thyroid cancer.

  19. Mixed lineage kinase-3/JNK1 axis promotes migration of human gastric cancer cells following gastrin stimulation.

    Science.gov (United States)

    Mishra, Prajna; Senthivinayagam, Subramanian; Rangasamy, Velusamy; Sondarva, Gautam; Rana, Basabi

    2010-03-01

    Gastrin is a gastrointestinal peptide hormone, secreted by the gastric G cells and can exist as a fully processed amidated form (G17) or as unprocessed forms. All forms of gastrin possess trophic properties towards the gastrointestinal mucosa. An understanding of the signaling pathways involved is important to design therapeutic approaches to target gastrin-mediated cellular events. The studies described here were designed to identify the signaling pathways by which amidated gastrin (G17) mediates cancer cell migration. These studies indicated a time- and dose-dependent increase in gastric cancer cell migration after G17 stimulation, involving cholecystokinin 2 receptor. G17-induced migration was preceded by activation of MAPK pathways and was antagonized after pretreatment with SP600125, a pharmacological inhibitor of c-Jun-NH(2)-terminal kinase (JNK) pathway. Knockdown of endogenous JNK1 expression via small interference RNA (JNK1-siRNA) inhibited G17-induced phosphorylation of c-Jun and migration, and overexpression of wild-type JNK1 or constitutive active JNK1 promoted G17-induced migration. Studies designed to identify the MAPK kinase kinase member mediating JNK activation indicated the involvement of mixed lineage kinase-3 (MLK3), which was transiently activated upon G17 treatment. Inhibition of MLK3 pathway via a pan-MLK inhibitor or knockdown of MLK3 expression by MLK3-siRNA antagonized G17-induced migration. Incubation with G17 also resulted in an induction of matrix metalloproteinase 7 promoter activity, which is known to mediate migration and invasion pathways in cancer cells. Modulation of MLK3, JNK1, and c-Jun pathways modulated G17-induced matrix metalloproteinase 7 promoter activation. These studies indicate that the MLK3/JNK1 axis mediates G17-induced gastric cancer cell migration, which can be targeted for designing novel therapeutic strategies for treating gastric malignancies.

  20. Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

    OpenAIRE

    Henderson, Jeffrey P.; Byun, Jaeman; Williams, Michelle V.; McCormick, Michael L.; Parks, William C.; Ridnour, Lisa A.; Heinecke, Jay W.

    2001-01-01

    Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by ...

  1. Downregulation of NIMA-related kinase-7 inhibits cell proliferation by inducing cell cycle arrest in human retinoblastoma cells.

    Science.gov (United States)

    Zhang, Jian; Wang, Li; Zhang, Yongkang

    2018-02-01

    NIMA-related kinase-7 (Nek7) is a centrosomal kinase involved in various types of cancer, including gallbladder cancer and hepatocellular carcinoma. However, the biological function and the potential underlying mechanism of Nek7 in retinoblastoma remain largely unknown. Therefore, the present study investigated the effects of Nek7 in retinoblastoma cells. The expression of Nek7 was initially determined and observed to be commonly upregulated in retinoblastoma cell lines (Y79, SO-RB50 and WERI-RB1) as compared with that in normal retinal pigment epithelium cells. Next, the endogenous expression of Nek7 was efficiently knocked down in Y79 and SO-RB50 cells using a lentivirus-mediated RNA interference approach, as confirmed by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Loss-of-function assays, including MTT, colony formation and flow cytometry, indicated that knockdown of Nek7 significantly inhibited cell growth, impaired the colony formation ability and induced cell cycle arrest at G0/G1 phase. Furthermore, mechanistic studies demonstrated that silencing of Nek7 resulted in reduced cyclin-dependent kinase 2, cyclin D1 and cyclin E levels in vitro . In conclusion, the present study highlights the crucial role of Nek7 in promoting retinoblastoma cell proliferation, and Nek7-silencing may serve as a novel therapeutic target for retinoblastoma.

  2. Effects of small interfering RNA inhibit Class I phosphoinositide 3-kinase on human gastric cancer cells

    Science.gov (United States)

    Zhu, Bao-Song; Yu, Li-Yan; Zhao, Kui; Wu, Yong-You; Cheng, Xiao-Li; Wu, Yong; Zhong, Feng-Yun; Gong, Wei; Chen, Qiang; Xing, Chun-Gen

    2013-01-01

    AIM: To investigate the effects of small interfering RNA (siRNA)-mediated inhibition of Class I phosphoinositide 3-kinase (Class I PI3K) signal transduction on the proliferation, apoptosis, and autophagy of gastric cancer SGC7901 and MGC803 cells. METHODS: We constructed the recombinant replication adenovirus PI3K(I)-RNA interference (RNAi)-green fluorescent protein (GFP) and control adenovirus NC-RNAi-GFP, and infected it into human gastric cancer cells. MTT assay was used to determine the growth rate of the gastric cancer cells. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after adenovirus PI3K(I)-RNAi-GFP and control adenovirus NC-RNAi-GFP treatment. Immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. The expression of autophagy was monitored with MDC, LC3 staining, and transmission electron microscopy. Western blotting was used to detect p53, Beclin-1, Bcl-2, and LC3 protein expression in the culture supernatant. RESULTS: The viability of gastric cancer cells was inhibited after siRNA targeting to the Class I PI3K blocked Class I PI3K signal pathway. MTT assays revealed that, after SGC7901 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 27.48% ± 2.71% at 24 h, 41.92% ± 2.02% at 48 h, and 50.85% ± 0.91% at 72 h. After MGC803 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 24.39% ± 0.93% at 24 h, 47.00% ± 0.87% at 48 h, and 70.30% ± 0.86% at 72 h (P p53 protein expression was significantly increased from 24 to 72 h. We also found that Bcl-2 protein expression down-regulated with the treatment of adenovirus PI3K(I)-RNAi-GFP (50 MOI). A number of isolated membranes, possibly derived from ribosome-free endoplasmic reticulum, were seen. These isolated membranes were elongated and

  3. The apoptotic mechanism of action of the sphingosine kinase 1 selective inhibitor SKI-178 in human acute myeloid leukemia cell lines.

    Science.gov (United States)

    Dick, Taryn E; Hengst, Jeremy A; Fox, Todd E; Colledge, Ashley L; Kale, Vijay P; Sung, Shen-Shu; Sharma, Arun; Amin, Shantu; Loughran, Thomas P; Kester, Mark; Wang, Hong-Gang; Yun, Jong K

    2015-03-01

    We previously developed SKI-178 (N'-[(1E)-1-(3,4-dimethoxyphenyl)ethylidene]-3-(4-methoxxyphenyl)-1H-pyrazole-5-carbohydrazide) as a novel sphingosine kinase-1 (SphK1) selective inhibitor and, herein, sought to determine the mechanism-of-action of SKI-178-induced cell death. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism of action of SKI-178 implicated c-Jun NH2-terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178-induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178-induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the prosurvival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multidrug resistance mediated by multidrug-resistant protein1 and/or prosurvival Bcl-2 family member overexpression did not affect the sensitivity of AML cells to SKI-178. Taken together, these findings highlight the therapeutic potential of SKI-178 targeting SphK1 as a novel therapeutic agent for the treatment of AML, including multidrug-resistant/recurrent AML subtypes. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  4. Methyl gallate isolated from Spondias pinnata exhibits anticancer activity against human glioblastoma by induction of apoptosis and sustained extracellular signal-regulated kinase 1/2 activation.

    Science.gov (United States)

    Chaudhuri, Dipankar; Ghate, Nikhil Baban; Singh, Sudhir Shankar; Mandal, Nripendranath

    2015-01-01

    Spondias pinnata has been reported for its efficient anticancer effects, but the studies were mostly focused on its extract. Since its bioactive compounds are largely unknown, this study was designed to characterize the lead components present in it and their anticancer activity against human glioblastoma cell line (U87). Major compounds from the ethyl acetate fraction were isolated by column chromatography and their anticancer potentials against U87 cells were evaluated. Furthermore, flow cytometric and immunoblotting analyses were performed to demonstrate the mechanism of apoptosis inducing activity of methyl gallate (MG) against U87 cell line. Four major compounds were isolated from the ethyl acetate fraction. Amongst these, two compounds showed promising activities and with the help of different spectroscopic methods they were identified as gallic acid and MG. Flow cytometric studies revealed that MG-induced apoptosis in U87 cells dose-dependently; the same was confirmed by activation of caspases through cleavage of endogenous substrate poly (adenosine diphosphate-ribose) polymerase. MG treatment also induced the expression of p53 and B-cell lymphoma-2-associated X and cleavage of BH3 interacting-domain with a concomitant decrease in B-cell lymphoma-2 expression. Moreover, MG-induced sustained phosphorylation of extracellular signal-regulated kinase (ERK1/2) in U87 cells with no change in the phosphorylation of other mitogen-activated protein kinases (c-Jun N-terminal of stress-activated protein kinases, p38). MG is a potent antioxidant and it induces sustained ERK1/2 activation and apoptosis in human glioblastoma U87, and provide a rationale for evaluation of MG for other brain carcinoma cell lines for the advancement of glioblastoma therapy.

  5. Transforming growth factor-β1 downregulates vascular endothelial growth factor-D expression in human lung fibroblasts via the Jun NH2-terminal kinase signaling pathway.

    Science.gov (United States)

    Cui, Ye; Osorio, Juan C; Risquez, Cristobal; Wang, Hao; Shi, Ying; Gochuico, Bernadette R; Morse, Danielle; Rosas, Ivan O; El-Chemaly, Souheil

    2014-03-20

    Vascular endothelial growth factor (VEGF)-D, a member of the VEGF family, induces both angiogenesis and lymphangiogenesis by activating VEGF receptor-2 (VEGFR-2) and VEGFR-3 on the surface of endothelial cells. Transforming growth factor (TGF)-β1 has been shown to stimulate VEGF-A expression in human lung fibroblast via the Smad3 signaling pathway and to induce VEGF-C in human proximal tubular epithelial cells. However, the effects of TGF-β1 on VEGF-D regulation are unknown. To investigate the regulation of VEGF-D, human lung fibroblasts were studied under pro-fibrotic conditions in vitro and in idiopathic pulmonary fibrosis (IPF) lung tissue. We demonstrate that TGF-β1 downregulates VEGF-D expression in a dose- and time-dependent manner in human lung fibroblasts. This TGF-β1 effect can be abolished by inhibitors of TGF-β type I receptor kinase and Jun NH2-terminal kinase (JNK), but not by Smad3 knockdown. In addition, VEGF-D knockdown in human lung fibroblasts induces G1/S transition and promotes cell proliferation. Importantly, VEGF-D protein expression is decreased in lung homogenates from IPF patients compared with control lung. In IPF lung sections, fibroblastic foci show very weak VEGF-D immunoreactivity, whereas VEGF-D is abundantly expressed within alveolar interstitial cells in control lung. Taken together, our data identify a novel mechanism for downstream signal transduction induced by TGF-β1 in lung fibroblasts, through which they may mediate tissue remodeling in IPF.

  6. Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

    Science.gov (United States)

    Janjusevic, Milijana; Greco, Stefania; Islam, Md Soriful; Castellucci, Clara; Ciavattini, Andrea; Toti, Paolo; Petraglia, Felice; Ciarmela, Pasquapina

    2016-11-01

    To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  7. Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, Anders; On, Stephen L. W.

    2007-01-01

    To investigate the bactericidal activity of antiviral and anticancer nucleoside analogues against a variety of pathogenic bacteria and characterize the activating enzymes, deoxyribonucleoside kinases (dNKs). Several FDA-approved nucleoside analogue drugs were screened for their potential...... bactericidal activity against several clinical bacterial isolates and type strains. We identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens...... phosphorylated AZT efficiently with a Km of 73.3 µM and kcat/Km of 6.6 × 10^4 s-1 M-1 and is the activator of this drug in vivo. 2",2"-Difluoro-2"-deoxycytidine (gemcitabine) was a potent antibiotic against Gram-positive bacteria in the concentration range between 0.001 and 1.0 µM. The B. cereus deoxyadenosine...

  8. Inhibition of Src family kinases overcomes anoikis resistance induced by spheroid formation and facilitates cisplatin-induced apoptosis in human mesothelioma cells.

    Science.gov (United States)

    Eguchi, Ryoji; Fujita, Yumiko; Tabata, Chiharu; Ogawa, Hiroyasu; Wakabayashi, Ichiro; Nakano, Takashi; Fujimori, Yoshihiro

    2015-11-01

    Malignant mesothelioma is an aggressive tumor arising from mesothelial cells of serous membranes, and forms spheroid-like cell aggregates in pleural and peritoneal effusions. We examined the levels of anoikis, apoptosis induced by the detachment of cells from the extracellur matrix, in suspension culture in the human mesothelioma cell line NCI-H2052. NCI-H2052 cells were adherent in conventional monolayer cultures, but were found to form spheroids in suspension cultures using dishes with ultra-low cell binding capacity. NCI-H2052 cells proliferated in both cultures, but the proliferation rate was markedly lower in suspension cultures than in monolayer cultures. In addition, NCI-H2052 cells in suspension cultures showed little apoptosis, suggesting that the suspension culture induces anoikis resistance. Western blot analysis revealed that suspension cultures induced activation of Src family kinases (SFK) after spheroid formation. Dasatinib, an inhibitor of multi-tyrosine kinases including SFK, abolished anoikis resistance in suspension cultures, indicating that SFK activated by spheroid formation are responsible for anoikis resistance. Cisplatin induced apoptosis in NCI-H2052 cells, but the apoptotic rate was significantly lower in suspension cultures than in monolayer cultures, suggesting that spheroid formation is involved in cisplatin resistance. Furthermore, a combination of dasatinib and cisplatin induced apoptosis more significantly than either alone in suspension cultures. These results suggest that spheroid formation induces resistance to anoikis and to cisplatin through SFK activation and that dasatinib facilitates cisplatin-induced apoptosis in human mesothelioma cells.

  9. Geldanamycin induces apoptosis in human gastric carcinomas by affecting multiple oncogenic kinases that have synergic effects with TNF-related apoptosis-inducing ligand.

    Science.gov (United States)

    Chen, Hui; Li, Liang-Qing; Pan, Dun

    2015-12-01

    The aim of the present study was to evaluate the effect of geldanamycin (GA) on the treatment of human gastric carcinomas and to investigate the molecular mechanism that provides the basis for the combination of GA with the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induction strategy. The expression of target proteins at the mRNA level was determined using reverse transcription-polymerase chain reaction (RT-PCR), and apoptosis was evaluated with the terminal deoxynucleotidyl transferase mediated digoxigenin-dUTP nick-end labeling and Annexin V/propidium iodide (PI) staining methods. Phosphorylation of targeted kinases was studied using immunocytochemistry methods, and malignant phenotypes were studied using in vitro assays. GA treatment inhibits proliferation, migration and invasion, and induces apoptosis in human gastric cancer SGC-7901 cells, most likely by decreasing the expression of B-RAF and by phosphorylation of protein kinase B (AKT) and ERK. The inhibitory role of AKT in TRAIL regulation holds considerable potential for achieving a synergic effect in clinical therapy, using a combination of GA treatment and TRAIL induction. The present study provides a basis for the future application of heat shock protein 90 (Hsp90) inhibitors, such as GA, in the clinical treatment of gastric cancer, particularly in combination therapies with TRAIL inducers.

  10. Campylobacter jejuni induces an anti-inflammatory response in human intestinal epithelial cells through activation of phosphatidylinositol 3-kinase/Akt pathway

    DEFF Research Database (Denmark)

    Li, Yiping; Vegge, Christina S.; Brøndsted, Lone

    2011-01-01

    to activate phosphatidylinositol 3-kinase (PI3K)/Akt pathway and induce pro-inflammatory interleukin-8(IL-8) as well as anti-inflammatory cytokine IL-10 in human intestinal epithelial cell line Colo 205. The signalling pathways PI3K/Akt and mitogen-activated protein (MAP)kinases ERK and p38 were involved in C....... jejuni-induced IL-8 and IL-10 expression. Inhibition of PI3K resulted in augmentation of C. jejuni-induced IL-8 production, concomitant with down-regulation of IL-10 mRNA, indicating an anti-inflammatory response was activated and associated with the activation of P13K/Akt. Similar effect was observed...... for cytolethal distending toxin (CDT) deficient mutants. Moreover, we demonstrated that heat-killed bacteria were able to induce IL-8 and IL-10 expression to a lower level than live bacteria. We therefore conclude that C. jejuni activate a PI3K/Akt-dependent anti-inflammatory pathway in human intestinal...

  11. Gene expression profiling of the synergy of 5-aza-2′-deoxycytidine and paclitaxel against renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Han Tiandong

    2012-09-01

    Full Text Available Abstract Background Renal cell carcinoma (RCC is one of the most common kidney cancers and is highly resistant to chemotherapy. We previously demonstrated that 5-aza-2′-deoxycytidine (DAC could significantly increase the susceptibility of renal cell carcinoma (RCC cells to paclitaxel (PTX treatment in vitro, and showed the synergy of DAC and PTX against RCC. The purpose of this study is to investigated the gene transcriptional alteration and investigate possible molecular mechanism and pathways implicated in the synergy of DAC and PTX against RCC. Methods cDNA microarray was performed and coupled with real-time PCR to identify critical genes in the synergistic mechanism of both agents against RCC cells. Various patterns of gene expression were observed by cluster analysis. IPA software was used to analyze possible biological pathways and to explore the inter-relationships between interesting network genes. Results We found that lymphoid enhancer-binding factor 1 (LEF1, transforming growth factor β-induced (TGFBI, C-X-C motif ligand 5 (CXCL5 and myelocytomatosis viral related oncogene (c-myc may play a pivotal role in the synergy of DAC and PTX. The PI3K/Akt pathway and other pathways associated with cyclins, DNA replication and cell cycle/mitotic regulation were also associated with the synergy of DAC and PTX against RCC. Conclusion The activation of PI3K/Akt-LEF1/β-catenin pathway could be suppressed synergistically by two agents and that PI3K/Akt-LEF1/β-catenin pathway is participated in the synergy of two agents.

  12. Equitoxic doses of 5-azacytidine and 5-aza-2'deoxycytidine induce diverse immediate and overlapping heritable changes in the transcriptome.

    Directory of Open Access Journals (Sweden)

    Xiangning Qiu

    2010-09-01

    Full Text Available The hypomethylating agent 5-Azacytidine (5-Aza-CR is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS. Surprisingly, the deoxyribonucleoside analog 5-Aza-2'deoxycytidine (5-Aza-CdR did not have a similar effect on survival in a large clinical trial. Both drugs are thought to exert their effects after incorporation into DNA by covalent binding of DNA methyltransferase (DNMT. While 5-Aza-CdR is incorporated into only DNA, 5-Aza-CR is also incorporated into RNA. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacities of these drugs to regulate the expression of mRNA and microRNAs (miRNA, which may potentially affect the activities of the drugs in patients.A hematopoietic (HL-60; acute myeloid leukemia and a solid (T24; transitional cell carcinoma cancer cell line were treated with equitoxic doses of 5-Aza-CR and 5-Aza-CdR for 24 hrs, and the immediate (day 2 and lasting (day 8 effects on RNA expression examined. There was considerable overlap between the RNAs heritably upregulated by both drugs on day 8 but more RNAs were stably induced by the deoxy analog. Both drugs strongly induced expression of cancer testis antigens. On day 2 more RNAs were downregulated by 5-Aza-CR, particularly at higher doses. A remarkable downregulation of miRNAs and a significant upregulation of tRNA synthetases and other genes involved in amino acid metabolism was observed in T24 cells.Overall, this suggests that significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping.

  13. Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Jia, Bingbing; Madsen, Lise; Petersen, Rasmus Koefoed

    2012-01-01

    Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA......) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence...... of the strong adipogenic inducers insulin, dexamethasone, and rosiglitazone, thereby clearly distinguishing the hMADS cells from murine preadipocytes cell lines, where rosiglitazone together with dexamethasone and insulin strongly promotes adipocyte differentiation. We further show that prostaglandin I(2) (PGI...

  14. 5'AMP activated protein kinase expression in human skeletal muscle: effects of strength training and type 2 diabetes

    DEFF Research Database (Denmark)

    Wojtaszewski, Jørgen; Birk, Jesper Bratz; Frøsig, Christian

    2005-01-01

    Strength training enhances insulin sensitivity and represents an alternative to endurance training for patients with type 2 diabetes (T2DM). The 5'AMP-activated protein kinase (AMPK) may mediate adaptations in skeletal muscle in response to exercise training; however, little is known about...... remained untrained (UT). Muscle biopsies were obtained before and after the training period. Basal AMPK activity and protein/mRNA expression of both catalytic (alpha1 and alpha2) and regulatory (beta1, beta2, gamma1, gamma2a, gamma2b and gamma3) AMPK isoforms were independent of T2DM, whereas the protein...

  15. Genetic Mapping and Functional Studies of a Natural Inhibitor of the Insulin Receptor Tyrosine Kinase: The Mouse Ortholog of Human α2-HS Glycoprotein

    Science.gov (United States)

    Cintrón, Vivian J.; Ko, Minoru S. H.; Chi, Kenneth D.; Gross, Jason P.; Srinivas, Pothur R.; Goustin, Anton Scott

    2000-01-01

    Fetuin/α2-HS glycoprotein (α2-HSG) homologs have been identified in several species including rat, sheep, pig, rabbit, guinea pig, cattle, mouse and human. Multiple physiological roles for these homologs have been suggested, including ability to bind to hydroxyapatite crystals and to specifically inhibit the tyrosine kinase (TK) activity of the insulin receptor (IR). In this study we report the identification, cloning, and characterization of the mouse Ahsg gene and its function as an IR-TK inhibitor. Genomic clones derived from a mouse Svj 129 genomic library were sequenced in order to characterize the intron–exon organization of the mouse Ahsg gene, including an 875 bp subclone containing 154 bp upstream from the transcription start site, the first exon, and part of the first intron. A second genomic subclone harboring a 3.45 kb Bgl II fragment contained exons 2, 3 and 4 in addition to two adjacent elements within the first intron-a repetitive element of the B1 family (92 bp) and a 271 bp tract of (T,C)n * (A,G)n. We have mapped mouse Ahsg at 16 cM adjacent to the Diacylglycerol kinase 3 (Dagk3) gene on chromosome 16 by genotyping interspecific backcross panels between C57BL/6J and Mus spretus. The position is syntenic with human chromosome 3q27, where the human AHSG gene resides. Using recombinant mouse α2-HSG expressed from a recombinant baculovirus, we demonstrate that mouse α2-HSG inhibits insulin–stimulated IR autophosphorylation and IR-TKA in vitro. In addition, mouse α2-HSG (25μg/ml) completely abolishes insulin-induced DNA synthesis in H-35 rat hepatoma cells. Based on the sequence data and functional analysis, we conclude that the mouse Ahsg gene is the true ortholog of the human AHSG gene. PMID:11467416

  16. Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

    Science.gov (United States)

    Henderson, Jeffrey P.; Byun, Jaeman; Williams, Michelle V.; McCormick, Michael L.; Parks, William C.; Ridnour, Lisa A.; Heinecke, Jay W.

    2001-01-01

    Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation. PMID:11172002

  17. A Rho-associated coiled-coil containing kinases (ROCK) inhibitor, Y-27632, enhances adhesion, viability and differentiation of human term placenta-derived trophoblasts in vitro.

    Science.gov (United States)

    Motomura, Kenichiro; Okada, Naoko; Morita, Hideaki; Hara, Mariko; Tamari, Masato; Orimo, Keisuke; Matsuda, Go; Imadome, Ken-Ichi; Matsuda, Akio; Nagamatsu, Takeshi; Fujieda, Mikiya; Sago, Haruhiko; Saito, Hirohisa; Matsumoto, Kenji

    2017-01-01

    Although human term placenta-derived primary cytotrophoblasts (pCTBs) represent a good human syncytiotrophoblast (STB) model, in vitro culture of pCTBs is not always easily accomplished. Y-27632, a specific inhibitor of Rho-associated coiled-coil containing kinases (ROCK), reportedly prevented apoptosis and improved cell-to-substrate adhesion and culture stability of dissociated cultured human embryonic stem cells and human corneal endothelial cells. The Rho kinase pathway regulates various kinds of cell behavior, some of which are involved in pCTB adhesion and differentiation. In this study, we examined Y-27632's potential for enhancing pCTB adhesion, viability and differentiation. pCTBs were isolated from term, uncomplicated placentas by trypsin-DNase I-Dispase II treatment and purified by HLA class I-positive cell depletion. Purified pCTBs were cultured on uncoated plates in the presence of epidermal growth factor (10 ng/ml) and various concentrations of Y-27632. pCTB adhesion to the plates was evaluated by phase-contrast imaging, viability was measured by WST-8 assay, and differentiation was evaluated by immunofluorescence staining, expression of fusogenic genes and hCG-β production. Ras-related C3 botulinum toxin substrate 1 (Rac1; one of the effector proteins of the Rho family) and protein kinase A (PKA) involvement was evaluated by using their specific inhibitors, NSC-23766 and H-89. We found that Y-27632 treatment significantly enhanced pCTB adhesion to plates, viability, cell-to-cell fusion and hCG-β production, but showed no effects on pCTB proliferation or apoptosis. Furthermore, NSC-23766 and H-89 each blocked the effects of Y-27632, suggesting that Y-27632 significantly enhanced pCTB differentiation via Rac1 and PKA activation. Our findings suggest that Rac1 and PKA may be interactively involved in CTB differentiation, and addition of Y-27632 to cultures may be an effective method for creating a stable culture model for studying CTB and STB biology

  18. Urea-induced denaturation of human calcium/calmodulin-dependent protein kinase IV: a combined spectroscopic and MD simulation studies.

    Science.gov (United States)

    Naz, Huma; Shahbaaz, Mohd; Haque, Md Anzarul; Bisetty, Krishna; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2017-02-01

    Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a multifunctional enzyme which belongs to the Ser/Thr kinase family. CaMKIV plays important role in varieties of biological processes such as gene expression regulation, memory consolidation, bone growth, T-cell maturation, sperm motility, regulation of microtubule dynamics, cell-cycle progression, and apoptosis. To measure stability parameters, urea-induced denaturation of CaMKIV was carried out at pH 7.4 and 25°C, using three different probes, namely far-UV CD, near-UV absorption, and tryptophan fluorescence. A coincidence of normalized denaturation curves of these optical properties suggests that urea-induced denaturation is a two-state process. Analysis of these denaturation curves gave values of 4.20 ± 0.12 kcal mol-1, 2.95 ± 0.15 M, and 1.42 ± 0.06 kcal mol-1 M-1 for [Formula: see text] (Gibbs free energy change (ΔGD) in the absence of urea), Cm (molar urea concentration ([urea]) at the midpoint of the denaturation curve), and m (=∂ΔGD/∂[urea]), respectively. All these experimental observations have been fully supported by 30 ns molecular dynamics simulation studies.

  19. Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells 1

    Science.gov (United States)

    Zeng, Fu-Yue; Dong, Hanqing; Cui, Jimmy; Liu, Lingling; Chen, Taosheng

    2009-01-01

    Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies. PMID:19995556

  20. High-resolution crystal structure of human Mapkap kinase 3 in complex with a high affinity ligand.

    Science.gov (United States)

    Cheng, Robert; Felicetti, Brunella; Palan, Shilpa; Toogood-Johnson, Ian; Scheich, Christoph; Barker, John; Whittaker, Mark; Hesterkamp, Thomas

    2010-01-01

    The Mapkap kinases 2 and 3 (MK2 and MK3) have been implicated in intracellular signaling pathways leading to the production of the pro-inflammatory cytokine tumor necrosis factor alpha. MK2 has been pursued by the biopharmaceutical industry for many years for the development of a small molecule anti-inflammatory treatment and drug-like inhibitors have been described. The development of some of these compounds, however, has been slowed by the absence of a high-resolution crystal structure of MK2. Herein we present a high-resolution (1.9 A) crystal structure of the highly homologous MK3 in complex with a pharmaceutical lead compound. While all of the canonical features of Ser/Thr kinases in general and MK2 in particular are recapitulated in MK3, the detailed analysis of the binding interaction of the drug-like ligand within the adenine binding pocket allows relevant conclusions to be drawn for the further design of potent and selective drug candidates.

  1. Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase

    Energy Technology Data Exchange (ETDEWEB)

    Filgueira de Azevedo, W. Jr.; Mueller-Dieckmann, H.J.; Schulze-Gahmen, U. [Lawrence Berkeley National Lab., CA (United States)] [and others

    1996-04-02

    The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl]-4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33-{Angstrom} resolution and refined to an R{sub factor} of 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2. 36 refs., 4 figs., 2 tabs.

  2. Identification of S664 TSC2 phosphorylation as a marker for extracellular signal-regulated kinase mediated mTOR activation in tuberous sclerosis and human cancer.

    Science.gov (United States)

    Ma, Li; Teruya-Feldstein, Julie; Bonner, Pauline; Bernardi, Rosa; Franz, David Neal; Witte, David; Cordon-Cardo, Carlos; Pandolfi, Pier Paolo

    2007-08-01

    Constitutive activation of extracellular signal-regulated kinases (Erk1/2) is frequently implicated in human cancers. Recently, aberrantly activated Erk was also found in brain lesions associated with tuberous sclerosis (TSC). We reported previously that Erk might contribute to tumorigenesis by phosphorylating TSC2 at specific residues, particularly S664. In our present study, 25 TSC-related cortical tubers or subependymal giant cell astrocytomas, as well as tissue microarrays of six types of human cancers, were analyzed for the expression of phospho-Erk (pErk) 1/2, S664-phospho-TSC2 (pTSC2), and phospho-S6 (pS6) by immunohistochemistry. We found that Erk-mediated TSC2 phosphorylation occurred at a high incidence and positively correlated with mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) activation in TSC-associated brain lesions as well as in various cancers. Interestingly, in certain types of cancers (e.g., breast carcinoma and colon carcinoma), S664-pTSC2 seemed to be a more sensitive marker than pErk. Furthermore, most of the pTSC2-positive samples ( approximately 75%) were positive for pS6, but only 40% to 55% of the pS6-positive tumors exhibited TSC2 phosphorylation. Our results show that S664 TSC2 phosphorylation is a marker for Erk-mediated (as opposed to Akt-mediated) mTOR activation in TSC and human cancer. On the basis of these findings, TSC2 phosphorylation at S664 can be used to identify patients that may benefit from antitumor therapy with MAPK and mTOR inhibitors. Importantly, our results indicate that Erk-mediated phosphorylation and inactivation of TSC2 can be critical in development of hamartomatous lesions in TSC and cancer pathogenesis.

  3. Casein Kinase 2 Is a Novel Regulator of the Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) Trafficking.

    Science.gov (United States)

    Chan, Ting; Cheung, Florence Shin Gee; Zheng, Jian; Lu, Xiaoxi; Zhu, Ling; Grewal, Thomas; Murray, Michael; Zhou, Fanfan

    2016-01-04

    Human organic anion transporting polypeptides (OATPs) mediate the influx of many important drugs into cells. Casein kinase 2 (CK2) is a critical protein kinase that phosphorylates >300 protein substrates and is dysregulated in a number of disease states. Among the CK2 substrates are several transporters, although whether this includes human OATPs has not been evaluated. The current study was undertaken to evaluate the regulation of human OATP1A2 by CK2. HEK-239T cells in which OATP1A2 was overexpressed were treated with CK2 specific inhibitors or transfected with CK2 specific siRNA, and the activity, expression, and subcellular trafficking of OATP1A2 was evaluated. CK2 inhibition decreased the uptake of the prototypic OATP1A2 substrate estrone-3-sulfate (E3S). Kinetic studies revealed that this was due to a decrease in the maximum velocity (Vmax) of E3S uptake, while the Michaelis constant was unchanged. The cell surface expression, but not the total cellular expression of OATP1A2, was impaired by CK2 inhibition and knockdown of the catalytic α-subunits of CK2. CK2 inhibition decreased the internalization of OATP1A2 via a clathrin-dependent pathway, decreased OATP1A2 recycling, and likely impaired OATP1A2 targeting to the cell surface. Consistent with these findings, CK2 inhibition also disrupted the colocalization of OATP1A2 and Rab GTPase (Rab)4-, Rab8-, and Rab9-positive endosomal and secretory vesicles. Taken together, CK2 has emerged as a novel regulator of the subcellular trafficking and stability of OATP1A2. Because OATP1A2 transports many molecules of physiological and pharmacological importance, the present data may inform drug selection in patients with diseases in which CK2 and OATP1A2 are dysregulated.

  4. Advanced glycation end products induce brain-derived neurotrophic factor release from human platelets through the Src-family kinase activation.

    Science.gov (United States)

    Furukawa, Kazuo; Fuse, Ichiro; Iwakura, Yuriko; Sotoyama, Hidekazu; Hanyu, Osamu; Nawa, Hiroyuki; Sone, Hirohito; Takei, Nobuyuki

    2017-02-08

    Brain-derived neurotrophic factor (BDNF) exerts beneficial effects not only on diabetic neuropathies but also on cardiovascular injury. There is argument regarding the levels of serum BDNF in patients with diabetes mellitus (DM). Because BDNF in peripheral blood is rich in platelets, this may represent dysregulation of BDNF release from platelets. Here we focused on advanced glycation end products (AGEs), which are elevated in patients with DM and have adverse effects on cardiovascular functions. The aim of this study is to elucidate the role of AGEs in the regulation of BDNF release from human platelets. Platelets collected from peripheral blood of healthy volunteers were incubated with various concentrations of AGE (glycated-BSA) at 37 °C for 5 min with or without BAPTA-AM, a cell permeable Ca(2+) chelator, or PP2, a potent inhibitor of Src family kinases (SFKs). Released and cellular BDNF were measured by ELISA and calculated. Phosphorylation of Src and Syk, a downstream kinase of SFKs, in stimulated platelets was examined by Western blotting and immunoprecipitation. AGE induced BDNF release from human platelets in a dose-dependent manner, which was dependent on intracellular Ca(2+) and SFKs. We found that AGE induced phosphorylation of Src and Syk. AGE induces BDNF release from human platelets through the activation of the Src-Syk-(possibly phospholipase C)-Ca(2+) pathway. Considering the toxic action of AGEs and the protective roles of BDNF, it can be hypothesized that AGE-induced BDNF release is a biological defense system in the early phase of diabetes. Chronic elevation of AGEs may induce depletion or downregulation of BDNF in platelets during the progression of DM.

  5. Synergistic antitumor effect of adenovirus armed with Drosophila melanogaster deoxyribonucleoside kinase and nucleoside analogs for human breast carcinoma in vitro and in vivo

    Science.gov (United States)

    Tang, Miao; Zu, Cong; He, Anning; Wang, Wenqian; Chen, Bo; Zheng, Xinyu

    2015-01-01

    Background Suicide gene therapy in cancer can selectively kill tumors without damaging normal tissues. Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK), an original suicide kinase, makes use of the carcinomatous suicide gene therapy for broader substrate specificity and a higher catalytic rate. Methods To enhance the anti-tumor efficacy of Dm-dNK and maintain its substrate specificity and safety control in the meantime, the conditionally replicative gene–viral system, ZD55–dNK (which contains the selective replication adenovirus, ZD55, encoded with Dm-dNK), was investigated in pushing a deeper development of this strategy. Selective replication, cell killing efficacy, and cytotoxicity, in combination with chemotherapy, were applied to two breast cell lines (MDA231 and MCF7 cells), two normal cell lines (WI38 and MRC5 cells), and the MCF7 xenograft model in vivo. Results The preclinical study showed that ZD55–dNK, combined with 2′,2′-difluorodeoxycytidine (DFDC), synergistically inhibited adenovirus replication in vitro but maintained specifically cancer cell killing efficacy. ZD55–dNK also greatly improved the antineoplastic effect in vitro and in breast cancer xenograft in vivo. Conclusion The concomitant use of ZD55–dNK and DFDC is possibly a novel and promising approach to breast cancer treatment, and further investigation on the safe control of excessive virus replication and the efficacy of this approach in humans is warranted. PMID:26203222

  6. Nintedanib modulates surfactant protein-D expression in A549 human lung epithelial cells via the c-Jun N-terminal kinase-activator protein-1 pathway.

    Science.gov (United States)

    Kamio, Koichiro; Usuki, Jiro; Azuma, Arata; Matsuda, Kuniko; Ishii, Takeo; Inomata, Minoru; Hayashi, Hiroki; Kokuho, Nariaki; Fujita, Kazue; Saito, Yoshinobu; Miya, Toshimichi; Gemma, Akihiko

    2015-06-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a high mortality rate. Signalling pathways activated by several tyrosine kinase receptors are known to be involved in lung fibrosis, and this knowledge has led to the development of the triple tyrosine kinase inhibitor nintedanib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), for the treatment of IPF. Pulmonary surfactant protein D (SP-D), an important biomarker of IPF, reportedly attenuates bleomycin-induced pulmonary fibrosis in mice. In this study, we investigated whether nintedanib modulates SP-D expression in human lung epithelial (A549) cells using quantitative real-time reverse transcriptase polymerase chain reaction and western blotting. To investigate the mechanisms underlying the effects of nintedanib, we evaluated the phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream target c-Jun. The effect of the JNK inhibitor SP600125 on c-Jun phosphorylation was also tested. Activation of activator protein-1 (AP-1) was examined using an enzyme-linked immunosorbent assay-based test, and cell proliferation assays were performed to estimate the effect of nintedanib on cell proliferation. Furthermore, we treated mice with nintedanib to examine its in vivo effect on SP-D levels in lungs. These experiments showed that nintedanib up-regulated SP-D messenger RNA expression in a dose-dependent manner at concentrations up to 5 μM, with significant SP-D induction observed at concentrations of 3 μM and 5 μM, in comparison with that observed in vehicle controls. Nintedanib stimulated a rapid increase in phosphorylated JNK in A549 cells within 30 min of treatment and stimulated c-Jun phosphorylation, which was inhibited by the JNK inhibitor SP600125. Additionally, nintedanib was found to activate AP-1. A549 cell proliferation was not affected by nintedanib at any of the tested

  7. Anti-VEGF strategies - from antibodies to tyrosine kinase inhibitors: background and clinical development in human cancer.

    LENUS (Irish Health Repository)

    Korpanty, Grzegorz

    2012-01-01

    Tumour angiogenesis (formation of new blood vessels supporting tumour growth and metastasis) is a result of complex interactions between the tumour and the surrounding microenvironment. Targeting tumours with anti-angiogenic therapy remains an exciting area of preclinical and clinical studies. Although many significant advances have been achieved and the clinical use of anti-angiogenic drugs is now well recognized in many solid malignancies, these therapies fall short of their anticipated clinical benefits and leave many unanswered questions like exact mechanism of action, patients\\' selection and monitoring response to anti-angiogenic drugs. Tumour angiogenesis is controlled by complex signaling cascades and ongoing research into molecular mechanisms of tumour angiogenesis not only helps to understand its basic mechanisms but hopefully will identify new therapeutic targets. In 2012, both monoclonal antibodies and small molecule tyrosine kinase inhibitors remain the two major clinically useful therapeutic options that interfere with tumour angiogenesis in many solid malignancies.

  8. Targeting elongation factor-2 kinase (eEF-2K) induces apoptosis in human pancreatic cancer cells.

    Science.gov (United States)

    Ashour, Ahmed A; Abdel-Aziz, Abdel-Aziz H; Mansour, Ahmed M; Alpay, S Neslihan; Huo, Longfei; Ozpolat, Bulent

    2014-01-01

    Pancreatic cancer (PaCa) is one of the most aggressive, apoptosis-resistant and currently incurable cancers with a poor survival rate. Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase, whose role in PaCa survival is not yet known. Here, we show that eEF-2K is overexpressed in PaCa cells and its down-regulation induces apoptotic cell death. Rottlerin (ROT), a polyphenolic compound initially identified as a PKC-δ inhibitor, induces apoptosis and autophagy in a variety of cancer cells including PaCa cells. We demonstrated that ROT induces intrinsic apoptosis, with dissipation of mitochondrial membrane potential (ΔΨm), and stimulates extrinsic apoptosis with concomitant induction of TNF-related apoptosis inducing ligand (TRAIL) receptors, DR4 and DR5, with caspase-8 activation, in PANC-1 and MIAPaCa-2 cells. Notably, while none of these effects were dependent on PKC-δ inhibition, ROT down-regulates eEF-2K at mRNA level, and induce eEF-2K protein degradation through ubiquitin-proteasome pathway. Down-regulation of eEF-2K recapitulates the events observed after ROT treatment, while its over-expression suppressed the ROT-induced apoptosis. Furthermore, eEF-2K regulates the expression of tissue transglutaminase (TG2), an enzyme previously implicated in proliferation, drug resistance and survival of cancer cells. Inhibition of eEF-2K/TG2 axis leads to caspase-independent apoptosis which is associated with induction of apoptosis-inducing factor (AIF). Collectively, these results indicate, for the first time, that the down-regulation of eEF-2K leads to induction of intrinsic, extrinsic as well as AIF-dependent apoptosis in PaCa cells, suggesting that eEF-2K may represent an attractive therapeutic target for the future anticancer agents in PaCa.

  9. Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro

    DEFF Research Database (Denmark)

    Engel, M; Issinger, O G; Lascu, I

    1994-01-01

    We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia...

  10. The mouse/human cross-species heterodimer of leucine-rich repeat kinase 2: possible significance in the transgenic model mouse of Parkinson's disease.

    Science.gov (United States)

    Miyajima, T; Ohta, E; Kawada, H; Maekawa, T; Obata, F

    2015-02-19

    Leucine-rich repeat kinase (LRRK2) is the causal molecule of autosomal dominant Parkinson's disease (PD). We previously reported that intracellular degradation of wild-type (WT) LRRK2 is promoted by formation of heterodimers with the I2020T mutant LRRK2. In the present study, we investigated whether this is also the case for mouse/human cross-species heterodimers, which could be formed in transgenic mice. First, by co-transfection and immunoprecipitation, we identified the cross-species heterodimer of mouse LRRK2 and human LRRK2. Next, we found that the protein level of mouse LRRK2 decreased when co-transfected with human I2020T LRRK2, but not with human WT LRRK2. These results suggested that degradation of mouse LRRK2 was promoted by formation of a cross-species heterodimer with the mutant LRRK2. In I2020T LRRK2-transgenic mice, the lower protein level of brain LRRK2 in comparison with control mice, together with higher expression of the mRNA, suggested that endogenous LRRK2 was degraded by formation of cross-species heterodimers. Our results suggest a new concept of cross-species dimer/oligomer formation in transgenic disease-model mice. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  11. JAK protein kinase inhibitors.

    Science.gov (United States)

    Thompson, James E

    2005-06-01

    In humans, the Janus protein tyrosine kinase family (JAKs) contains four members: JAK1, JAK2, JAK3 and TYK2. JAKs phosphorylate signal transducers and activators of transcription (STATs) simultaneously with other phosphorylations required for activation, and there are several cellular mechanisms in place to inhibit JAK/STAT signaling. That one might be able to modulate selected JAK/STAT-mediated cellular signals by inhibiting JAK kinase activity to effect a positive therapeutic outcome is a tantalizing prospect, as yet incompletely realized. While current data suggest no therapeutic use for JAK1 and TYK2 inhibition, JAK2 inhibition seems a promising but not definitively tested mechanism for treatment of leukemia. More promising, however, are data indicating a possible therapeutic use of JAK3 inhibition. The restriction of the JAK3-deficient phenotype to the hematopoietic system and the resulting profound immune suppression suggest that JAK3 could be a target for immunosuppressive therapies used to prevent organ transplant rejection.

  12. TGFβ activated kinase 1 (TAK1 at the crossroad of B cell receptor and Toll-like receptor 9 signaling pathways in human B cells.

    Directory of Open Access Journals (Sweden)

    Dániel Szili

    Full Text Available B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR, receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R and the innate receptor, Toll-like receptor 9 (TLR9. However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs, ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1 is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.

  13. Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-βI and prooxidant enzyme NADPH oxidase

    Directory of Open Access Journals (Sweden)

    Beili Shao

    2014-01-01

    Full Text Available Blood–brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM significantly increased NADPH oxidase activity, O2•− generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-α, PKC-β or PKC-βI via their specific inhibitors and neutralisation of O2•− by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-βI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2•− production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-βI and NADPH oxidase.

  14. The cell division control protein 42-Src family kinase-neural Wiskott-Aldrich syndrome protein pathway regulates human proplatelet formation.

    Science.gov (United States)

    Palazzo, A; Bluteau, O; Messaoudi, K; Marangoni, F; Chang, Y; Souquere, S; Pierron, G; Lapierre, V; Zheng, Y; Vainchenker, W; Raslova, H; Debili, N

    2016-12-01

    Essentials The role of the cytoskeleton during megakaryocyte differentiation was examined. Human megakaryocytes are derived from in vitro cultured CD34(+) cells. Cell division control protein 42 (CDC42) positively regulates proplatelet formation (PPF). Neural Wiskott-Aldrich syndrome protein, the main effector of CDC42 with Src positively regulates PPF. Background Cytoskeletal rearrangements are essential for platelet release. The RHO small GTPase family, as regulators of the actin cytoskeleton, play an important role in proplatelet formation (PPF). In the neuronal system, CDC42 is involved in axon formation, a process that combines elongation and branching as for PPF. Objective To analyze the role of CDC42 and its effectors of the Wiskott-Aldrich syndrome protein (WASP) family in PPF. Methods Human megakaryocytes (MKs) were obtained from CD34(+) cells. Inhibition of CDC42 in MKs was performed with the chemical inhibitor CASIN or with an active or a dominant-negative form of CDC42. The knock-down of N-WASP was obtained with a small hairpin RNA strategy Results Herein, we show that CDC42 activity increased during MK differentiation. The use of the chemical inhibitor CASIN or of an active or a dominant-negative form of CDC42 demonstrated that CDC42 positively regulated PPF in vitro. We determined that N-WASP, but not WASP, regulated PPF. We found that N-WASP knockdown led to a marked decrease in PPF, owing to a defect in the demarcation membrane system (DMS). This was associated with RHOA activation, and a concomitant augmentation in the phosphorylation of mysosin light chain 2. Phosphorylation of N-WASP, creating a primed form of N-WASP, increased during MK differentiation. Phosphorylation inhibition by two Src family kinase inhibitors decreased PPF. Conclusions We conclude that N-WASP positively regulates DMS development and PPF, and that the Src family kinases in association with CDC42 regulate PPF through N-WASP. © 2016 International Society on Thrombosis and

  15. Danusertib, a potent pan-Aurora kinase and ABL kinase inhibitor, induces cell cycle arrest and programmed cell death and inhibits epithelial to mesenchymal transition involving the PI3K/Akt/mTOR-mediated signaling pathway in human gastric cancer AGS and NCI-N78 cells.

    Science.gov (United States)

    Yuan, Chun-Xiu; Zhou, Zhi-Wei; Yang, Yin-Xue; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Tianxing; Pan, Si-Yuan; Chen, Xiao-Wu; Zhou, Shu-Feng

    2015-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide, with a poor response to current chemotherapy. Danusertib is a pan-inhibitor of the Aurora kinases and a third-generation Bcr-Abl tyrosine kinase inhibitor with potent anticancer effects, but its antitumor effect and underlying mechanisms in the treatment of human gastric cancer are unknown. This study aimed to investigate the effects of danusertib on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition and the molecular mechanisms involved in human gastric cancer AGS and NCI-N78 cells. The results showed that danusertib had potent growth-inhibitory, apoptosis-inducing, and autophagy-inducing effects on AGS and NCI-N78 cells. Danusertib arrested AGS and NCI-N78 cells in G2/M phase, with downregulation of expression of cyclin B1 and cyclin-dependent kinase 1 and upregulation of expression of p21 Waf1/Cip1, p27 Kip1, and p53. Danusertib induced mitochondria-mediated apoptosis, with an increase in expression of proapoptotic protein and a decrease in antiapoptotic proteins in both cell lines. Danusertib induced release of cytochrome c from the mitochondria to the cytosol and triggered activation of caspase 9 and caspase 3 in AGS and NCI-N78 cells. Further, danusertib induced autophagy, with an increase in expression of beclin 1 and conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) to LC3-II in both cell lines. Inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways as well as activation of 5' AMP-activated protein kinase contributed to the proautophagic effect of danusertib in AGS and NCI-N78 cells. SB202191 and wortmannin enhanced the autophagy-inducing effect of danusertib in AGS and NCI-N78 cells. In addition, danusertib inhibited epithelial to mesenchymal transition with an increase in expression of E-cadherin and a decrease in expression of

  16. Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway.

    Science.gov (United States)

    Hara, Tomoya; Kakudo, Natsuko; Morimoto, Naoki; Ogawa, Takeshi; Lai, Fangyuan; Kusumoto, Kenji

    2016-12-01

    Platelet-rich plasma (PRP) contains a high concentration of several growth factors and contributes to soft-tissue engineering and wound healing. However, the effect of PRP on human dermal fibroblast proliferation and responses is unknown. This was investigated in the present study using PRP prepared from the whole human blood using the double-spin method. Human dermal fibroblast cultures were established from skin samples collected during plastic surgery. Platelet concentration and growth factor levels in PRP were estimated, and a cell proliferation assay was carried out after PRP treatment. The role of Ras-dependent extracellular signal-regulated kinase (ERK)1/2 in the effects of PRP was investigated in human dermal fibroblasts by suppressing ERK1/2 expression with an inhibitor or by short interfering (si)RNA-mediated knockdown, and assessing ERK1/2 phosphorylation by western blotting as well as proliferation in PRP-treated cells. We found that PRP stimulated human dermal fibroblast proliferation, which was suppressed by ERK1/2 inhibitor treatment (P < 0.01). ERK1/2 phosphorylation was increased in the presence of PRP, while siRNA-mediated knockdown of ERK1/2 blocked cell proliferation normally induced by PRP treatment (P < 0.01). These results demonstrate that PRP induces human dermal fibroblast proliferation via activation of ERK1/2 signaling. Our findings provide a basis for the development of agents that can promote wound healing and can be applied to soft-tissue engineering.

  17. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

    Directory of Open Access Journals (Sweden)

    Li JP

    2015-02-01

    , but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl and B-cell lymphoma 2 (Bcl-2, but increased the expression of Bcl-2-associated X protein (Bax and p53-upregulated modulator of apoptosis (PUMA, and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK and extracellular signal-regulated kinases 1 and 2 (Erk1/2 and inhibited the activation of protein kinase B (Akt/mammalian target of rapamycin (mTOR signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition and small interfering RNA-mediated knockdown of Erk1/2 also remarkably increased the level of LC3-II in MCF7 cells. Moreover, Danu inhibited EMT in both MCF7 and MDA-MB-231 cells with upregulated E

  18. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

    Directory of Open Access Journals (Sweden)

    Cong Haolong

    Full Text Available Enterovirus 71 (EV71 is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  19. Enterovirus 71 VP1 Activates Calmodulin-Dependent Protein Kinase II and Results in the Rearrangement of Vimentin in Human Astrocyte Cells

    Science.gov (United States)

    Haolong, Cong; Du, Ning; Hongchao, Tian; Yang, Yang; Wei, Zhang; Hua, Zhang; Wenliang, Zhang; Lei, Song; Po, Tien

    2013-01-01

    Enterovirus 71 (EV71) is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II) which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human. PMID:24073199

  20. Effects of sodium lactate Ringer's injection on transfection of human protein kinase C-α antisense oligonucleotide in A549 lung cancer cells.

    Science.gov (United States)

    Wang, Z H; Sun, W W; Han, Y L; Ma, Z

    2016-08-26

    In the present study, we evaluated the effects of four solutions [Dulbecco's modified Eagle's medium (DMEM), sodium lactate Ringer's injection (SLRI), phosphate-buffered saline (PBS), and NaCl] on the transfection of the human protein kinase C-a antisense oligonucleotide (PKC-a ASO) aprinocarsen in human lung carcinoma A549 cells. Specifically, SLRI, DMEM, PBS, or NaCl were used as the growth solutions for A549 cells, and OPTI-MEM was used as the PKC-a ASO diluent for transfection. Additionally, SLRI, DMEM, PBS, or NaCl were used as both the growth solutions and diluents for transfection. The cell viability and transfection efficiency were determined. The results demonstrated that when SLRI was used as either the growth solution or both the growth solution and diluent for aprinocarsen transfection in A549 cells, the effects were close to the best effects observed with DMEM as the growth solution and OPTI-MEM as the diluent, which supported the transfection of aprinocarsen into the cells. Moreover, SLRI resulted in higher transfection efficiency than those of PBS and NaCl. In in vitro experiments, aprinocarsen effectively induced apoptosis in A549 cells. In conclusion, SLRI may replace PBS or NaCl in clinical trials as a transfection solution readily accepted by the human body. To our knowledge, this is the first report demonstrating the use of SLRI as a transfection solution in lung-cancer cell lines.

  1. The Natural Stilbenoid Piceatannol Decreases Activity and Accelerates Apoptosis of Human Neutrophils: Involvement of Protein Kinase C

    Directory of Open Access Journals (Sweden)

    Viera Jancinova

    2013-01-01

    Full Text Available Neutrophils are able to release cytotoxic substances and inflammatory mediators, which, along with their delayed apoptosis, have a potential to maintain permanent inflammation. Therefore, treatment of diseases associated with chronic inflammation should be focused on neutrophils; formation of reactive oxygen species and apoptosis of these cells represent two promising targets for pharmacological intervention. Piceatannol, a naturally occurring stilbenoid, has the ability to reduce the toxic action of neutrophils. This substance decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. Radicals formed within neutrophils (fulfilling a regulatory role were reduced to a lesser extent than extracellular oxidants, potentially dangerous for host tissues. Moreover, piceatannol did not affect the phosphorylation of p40phox—a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular (granular membranes. The stilbenoid tested elevated the percentage of early apoptotic neutrophils, inhibited the activity of protein kinase C (PKC—the main regulatory enzyme in neutrophils, and reduced phosphorylation of PKC isoforms α, βII, and δ on their catalytic region. The results indicated that piceatannol may be useful as a complementary medicine in states associated with persisting neutrophil activation and with oxidative damage of tissues.

  2. Lysophosphatidic acid induces reactive oxygen species generation by activating protein kinase C in PC-3 human prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Chu-Cheng; Lin, Chuan-En; Lin, Yueh-Chien [Institute of Zoology, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Ju, Tsai-Kai [Instrumentation Center, National Taiwan University, Taipei, Taiwan, ROC (China); Technology Commons, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Huang, Yuan-Li [Department of Biotechnology, Asia University, Taichung, Taiwan, ROC (China); Lee, Ming-Shyue [Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC (China); Chen, Jiun-Hong [Institute of Zoology, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Department of Life Science, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Lee, Hsinyu, E-mail: hsinyu@ntu.edu.tw [Institute of Zoology, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Department of Life Science, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Center for Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan, ROC (China)

    2013-11-01

    Highlights: •LPA induces ROS generation through LPA{sub 1} and LPA{sub 3}. •LPA induces ROS generation by activating PLC. •PKCζ mediates LPA-induced ROS generation. -- Abstract: Prostate cancer is one of the most frequently diagnosed cancers in males, and PC-3 is a cell model popularly used for investigating the behavior of late stage prostate cancer. Lysophosphatidic acid (LPA) is a lysophospholipid that mediates multiple behaviors in cancer cells, such as proliferation, migration and adhesion. We have previously demonstrated that LPA enhances vascular endothelial growth factor (VEGF)-C expression in PC-3 cells by activating the generation of reactive oxygen species (ROS), which is known to be an important mediator in cancer progression. Using flow cytometry, we showed that LPA triggers ROS generation within 10 min and that the generated ROS can be suppressed by pretreatment with the NADPH oxidase (Nox) inhibitor diphenylene iodonium. In addition, transfection with LPA{sub 1} and LPA{sub 3} siRNA efficiently blocked LPA-induced ROS production, suggesting that both receptors are involved in this pathway. Using specific inhibitors and siRNA, phospholipase C (PLC) and protein kinase C (PKC) were also suggested to participate in LPA-induced ROS generation. Overall, we demonstrated that LPA induces ROS generation in PC-3 prostate cancer cells and this is mediated through the PLC/PKC/Nox pathway.

  3. Hub nodes in the network of human Mitogen-Activated Protein Kinase (MAPK pathways: Characteristics and potential as drug targets

    Directory of Open Access Journals (Sweden)

    V.K. MD Aksam

    2017-01-01

    Full Text Available Proteins involved in the cross-talk between ERK1/2, ERK5, JNK, and P38 signalling pathways integrate the network of Mitogen-Activated Protein Kinase (MAPK pathways. Graph theory-based approach is used to construct the network of MAPK pathways, and to observe the network organisational principles. Connectivity pattern reveals rich-club among the hubs, enabling structural ordering. A positive correlation between the degree of the nodes and percentage of essential protein showed hubs are central to the network architecture and function. Furthermore, attributes like connectivity, inter/intra-pathway class, position in the pathway, protein type and subcellular localization of the essential and non-essential proteins are characterizing complex functional roles. Shared properties of 34 cancerous essential proteins lack to be drug targets. We identified the seven nodes overlapping properties of the hub, essential and causing side effects on targeting them. We exploit the strategy of cancerous, non-hub and non-essential proteins as potential drug targets and identified 4EBP1, BAD, CHOP10, GADD45, HSP27, MKP1, RNPK, MLTKa/b, cPLA2, eEF2K and elF4E. We have illustrated the implication of targeting hub nodes and proposed network-based drug targets which would cause less side effect.

  4. Exploring the interaction between human focal adhesion kinase and inhibitors: a molecular dynamic simulation and free energy calculations.

    Science.gov (United States)

    Zhan, Jiu-Yu; Zhang, Ji-Long; Wang, Yan; Li, Ye; Zhang, Hong-Xing; Zheng, Qing-Chuan

    2016-11-01

    Focal adhesion kinase is an important target for the treatment of many kinds of cancers. Inhibitors of FAK are proposed to be the anticancer agents for multiple tumors. The interaction characteristic between FAK and its inhibitors is crucial to develop new inhibitors. In the present article, we used Molecular Dynamic (MD) simulation method to explore the characteristic of interaction between FAK and three inhibitors (PHM16, TAE226, and ligand3). The MD simulation results together with MM-GB/SA calculations show that the combinations are enthalpy-driven process. Cys502 and Asp564 are both essential residues due to the hydrogen bond interactions with inhibitors, which was in good agreement with experimental data. Glu500 can form a non-classical hydrogen bond with each inhibitor. Arg426 can form electrostatic interactions with PHM16 and ligand3, while weaker with TAE226. The electronic static potential was employed, and we found that the ortho-position methoxy of TAE226 has a weaker negative charge than the meta-position one in PHM16 or ligand3. Ile428, Val436, Ala452, Val484, Leu501, Glu505, Glu506, Leu553, Gly563 Leu567, Ser568 are all crucial residues in hydrophobic interactions. The key residues in this work will be available for further inhibitor design of FAK and also give assistance to further research of cancer.

  5. MHC class I ligation of human T cells activates the ZAP70 and p56lck tyrosine kinases, leads to an alternative phenotype of the TCR/CD3 zeta-chain, and induces apoptosis

    DEFF Research Database (Denmark)

    Skov, S; Bregenholt, S; Claesson, Mogens Helweg

    1997-01-01

    Cross-linking of MHC class I (MHC-I) molecules on human T cells induces signal-transduction events, including activation of tyrosine kinases, tyrosine phosphorylation of phospholipase C-gamma 1, and elevation of the intracellular free calcium concentration. In this study, we demonstrate that the ...

  6. Acute hypersensitivity of pluripotent testicular cancer-derived embryonal carcinoma to low-dose 5-aza deoxycytidine is associated with global DNA Damage-associated p53 activation, anti-pluripotency and DNA demethylation.

    Directory of Open Access Journals (Sweden)

    Bijesh K Biswal

    Full Text Available Human embryonal carcinoma (EC cells are the stem cells of nonseminoma testicular germ cells tumors (TGCTs and share remarkable similarities to human embryonic stem (ES cells. In prior work we found that EC cells are hypersensitive to low nanomolar doses of 5-aza deoxycytidine (5-aza and that this hypersensitivity partially depended on unusually high levels of the DNA methyltransferase, DNMT3B. We show here that low-dose 5-aza treatment results in DNA damage and induction of p53 in NT2/D1 cells. In addition, low-dose 5-aza results in global and gene specific promoter DNA hypomethylation. Low-dose 5-aza induces a p53 transcriptional signature distinct from that induced with cisplatin in NT2/D1 cells and also uniquely downregulates genes associated with pluripotency including NANOG, SOX2, GDF3 and Myc target genes. Changes in the p53 and pluripotency signatures with 5-aza were to a large extent dependent on high levels of DNMT3B. In contrast to the majority of p53 target genes upregulated by 5-aza that did not show DNA hypomethylation, several other genes induced with 5-aza had corresponding decreases in promoter methylation. These genes include RIN1, SOX15, GPER, and TLR4 and are novel candidate tumors suppressors in TGCTs. Our studies suggest that the hypersensitivity of NT2/D1 cells to low-dose 5-aza is multifactorial and involves the combined activation of p53 targets, repression of pluripotency genes, and activation of genes repressed by DNA methylation. Low-dose 5-aza therapy may be a general strategy to treat those tumors that are sustained by cells with embryonic stem-like properties.GEO NUMBER FOR THE MICROARRAY DATA: GSE42647.

  7. Characterization and mapping of the human rhodopsin kinase gene and screening of the gene for mutations in patients with retinitis pigmentosa

    Energy Technology Data Exchange (ETDEWEB)

    Khani, S.C.; Lin, D.; Magovcevic, I. [Harvard Medical School, Boston, MA (United States)] [and others

    1994-09-01

    Rhodopsin kinase (RK) is a cytosolic enzyme in rod photoreceptors that initiates the deactivation of the phototransductions cascade by phosphorylating photoactivated rhodopsin. Although the cDNA sequence of bovine RK has been determined previously, no human cDNA or genomic sequence has thus far been available for genetic studies. In order to investigate the possible role of this candidate gene in retinitis pigmentosa (RP) and allied diseases, we have isolated and characterized human cDNA and genomic clones derived from the RK locus. The coding sequence of the human gene is 1692 nucleotides in length and is split into seven exons. The human and the bovine sequence show 84% identity at the nucleotide level and 92% identity at the amino acid level. Thus far, the intronic sequences flanking each exon except for one have been determined. We have also mapped the human RK gene to chromosome 13q34 using fluorescence in situ hybridization. To our knowledge, no RP gene has as yet been linked to this region. However, since the substrate for RK (rhodopsin) and other members of the phototransduction cascade have been implicated in the pathogenesis of RP, it is conceivable that defects in RK can also cause some forms of this disease. We are evaluating this possibility by screening DNA from 173 patients with autosomal recessive RP and 190 patients with autosomal dominant RP. So far, we have found 11 patients with variant bands. In one patient with autosomal dominant RP we discovered the missense change Ser536Leu. Cosegregation studies and further sequencing of the variant bands are currently underway.

  8. Role of protein kinase A in the inhibition of human mast cell histamine release by β-adrenergic receptor agonists

    Directory of Open Access Journals (Sweden)

    Toshinobu Kato

    2002-01-01

    Conclusions: The present results suggest that PKA activation induced by β-adrenergic receptor agonists plays a crucial role in inhibiting IgE-mediated histamine release from human cultured mast cells through suppressing PKC translocation.

  9. Involvement of kinase PKC-zeta in the p62/p62(P392L)-driven activation of NF-κB in human osteoclasts.

    Science.gov (United States)

    Chamoux, Estelle; McManus, Stephen; Laberge, Gino; Bisson, Martine; Roux, Sophie

    2013-03-01

    Mutations of the gene encoding sequestosome1 (SQSTM1/p62), clustering in or near the UBA domain, have been described in Paget's disease of bone (PDB); among these the P392L substitution is the most prevalent. Protein p62 mediates several cell functions, including the control of NF-κB signaling, and autophagy. This scaffolding protein interacts with atypical PKCζ in the RANKL-induced signaling complex. We have previously shown that osteoclasts (OCs) overexpressing the p62(P392L) variant were in a constitutively activated state, presenting activated kinase p-PKCζ/λ and activated NF-κB prior to RANKL stimulation. In the present study, we investigated the relationships between PKCζ and NF-κB activation in human OCs transfected with p62 variants. We showed that PKCζ and p-PKCζ/λ co-localize with p62, and that PKCζ is involved in the RANKL-induced NF-κB activation and in the RANKL-independent activation of NF-κB observed in p62(P392L)-transfected cells. We also observed a basal and RANKL-induced increase in IκBα levels in the presence of the p62(P392L) mutation that contrasted with the NF-κB activation. In this study we propose that PKCζ plays a role in the activation of NF-κB by acting as a p65 (RelA) kinase at Ser(536), independently of IκBα; this alternative pathway could be used preferentially in the presence of the p62(P392L) mutation, which may hinder the ubiquitin-proteasome pathway. Overall, our results highlight the importance of p62-associated PKCζ in the overactive state of pagetic OCs and in the activation of NF-κB, particularly in the presence of the p62(P392L) mutation. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Synergistic antitumor effect of adenovirus armed with Drosophila melanogaster deoxyribonucleoside kinase and nucleoside analogs for human breast carcinoma in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Tang M

    2015-07-01

    Full Text Available Miao Tang,1 Cong Zu,2 Anning He,2 Wenqian Wang,1 Bo Chen,1 Xinyu Zheng1,2 1Department of Breast Surgery, The First Hospital of China Medical University, 2Laboratory 1, Cancer Institute, China Medical University, Shenyang, People’s Republic of China Background: Suicide gene therapy in cancer can selectively kill tumors without damaging normal tissues. Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK, an original suicide kinase, makes use of the carcinomatous suicide gene therapy for broader substrate specificity and a higher catalytic rate.Methods: To enhance the anti-tumor efficacy of Dm-dNK and maintain its substrate specificity and safety control in the meantime, the conditionally replicative gene–viral system, ZD55–dNK (which contains the selective replication adenovirus, ZD55, encoded with Dm-dNK, was investigated in pushing a deeper development of this strategy. Selective replication, cell killing efficacy, and cytotoxicity, in combination with chemotherapy, were applied to two breast cell lines (MDA231 and MCF7 cells, two normal cell lines (WI38 and MRC5 cells, and the MCF7 xenograft model in vivo.Results: The preclinical study showed that ZD55–dNK, combined with 2',2'-difluorodeoxycytidine (DFDC, synergistically inhibited adenovirus replication in vitro but maintained specifically cancer cell killing efficacy. ZD55–dNK also greatly improved the antineoplastic effect in vitro and in breast cancer xenograft in vivo.Conclusion: The concomitant use of ZD55–dNK and DFDC is possibly a novel and promising approach to breast cancer treatment, and further investigation on the safe control of excessive virus replication and the efficacy of this approach in humans is warranted. Keywords: Dm-dNK, oncolytic adenovirus, cancer suicide gene therapy, nucleoside analogs, safety control

  11. 20(S)-Protopanaxadiol induces apoptosis in human hepatoblastoma HepG2 cells by downregulating the protein kinase B signaling pathway.

    Science.gov (United States)

    Lu, Zeyuan; Xu, Huali; Yu, Xiaofeng; Wang, Yuchen; Huang, Long; Jin, Xin; Sui, Dayun

    2018-02-01

    Hepatoblastoma is the most common primary liver tumor for children aged S)-Protopanaxadiol (PPD) is a ginsenoside extracted from Pananx quinquefolium L ., which inhibits tumor growth in several cancer cell lines. The purpose of the present study was to assess the anticancer activities of 20(S)-PPD in human hepatoblastoma HepG2 cells. The cytotoxicity of 20(S)-PPD on HepG2 cells was evaluated using an MTT assay. Apoptosis was detected using DAPI staining and flow cytometry. The expression of apoptosis-associated proteins was identified by western blotting. The results demonstrated that 20(S)-PPD inhibited the viability of HepG2 cell in a dose and time-dependent manner. The IC 50 values were 81.35, 73.5, 48.79 µM at 24, 48 and 72 h, respectively. Topical morphological changes of apoptotic body formation following 20(S)-PPD treatment were detected by DAPI staining. The percentage of Annexin V-fluoroscein isothyiocyanate positive cells were 3.73, 17.61, 23.44 and 65.43% in HepG2 cells treated with 0, 40, 50 and 60 µM of 20(S)-PPD, respectively. Furthermore, 20(S)-PPD upregulated the expression of Bax and downregulated the expression of Bcl-2 and also activated caspases-3 and -9, and Poly [ADP-ribose] polymerase cleavage. In addition, 20(S)-PPD inhibited the phosphorylation of protein kinase B (Akt; Ser473). The results indicate that 20(S)-PPD inhibits the viability of HepG2 cells and induces apoptosis in HepG2 cells by inhibiting the phosphoinositide-3-kinase/Akt pathway.

  12. Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells in Phenotypic Screening: A Transforming Growth Factor-β Type 1 Receptor Kinase Inhibitor Induces Efficient Cardiac Differentiation.

    Science.gov (United States)

    Drowley, Lauren; Koonce, Chad; Peel, Samantha; Jonebring, Anna; Plowright, Alleyn T; Kattman, Steven J; Andersson, Henrik; Anson, Blake; Swanson, Bradley J; Wang, Qing-Dong; Brolen, Gabriella

    2016-02-01

    Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium, functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts, we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system, these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs, we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust, scalable, and consistent methodology. In the present study, we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set, we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality, with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic

  13. Angiotensin II activates MAP kinase and NF-kappaB through angiotensin II type I receptor in human pancreatic cancer cells.

    Science.gov (United States)

    Amaya, Koji; Ohta, Tetsuo; Kitagawa, Hirohisa; Kayahara, Masato; Takamura, Hiroyuki; Fujimura, Takashi; Nishimura, Gen-Ichi; Shimizu, Koichi; Miwa, Koichi

    2004-10-01

    Pancreatic ductal cancer has higher angiotensin II concentrations compared with normal pancreas or other solid tumors. This study examined angiotensin II type 1 (AT1) receptor expression and the role of angiotensin II in proliferation and survival of human pancreatic cancer cells. All three pancreatic cancer cell lines studied, from well to poorly-differentiated types, HPAF-II, AsPC-1, and Panc-1, showed strong expression of AT1 receptor. In contrast, HT-29 human colon cancer cells showed extremely weak expression. Angiotensin II stimulated the growth of pancreatic cancer cells through MAP kinase activation but had no significant effect on proliferation of HT-29 colon cancer cells. In addition, angiotensin II significantly prevented cisplatin (CDDP)-induced apoptosis through NF-kappaB activation and the subsequent production of anti-apoptotic molecules, including survivin and Bcl-XL, in pancreatic cancer cells. These findings suggest that angiotensin II plays a role in the growth and chemoresistance of AT1-positive pancreatic cancer cells through its action as a potent mitogen and anti-apoptotic molecule.

  14. An Active Form of Sphingosine Kinase-1 Is Released in the Extracellular Medium as Component of Membrane Vesicles Shed by Two Human Tumor Cell Lines

    Directory of Open Access Journals (Sweden)

    Salvatrice Rigogliuso

    2010-01-01

    Full Text Available Expression of sphingosine kinase-1 (SphK-1 correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P, a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis.

  15. Citron kinase - renaissance of a neglected mitotic kinase.

    Science.gov (United States)

    D'Avino, Pier Paolo

    2017-05-15

    Cell division controls the faithful segregation of genomic and cytoplasmic materials between the two nascent daughter cells. Members of the Aurora, Polo and cyclin-dependent (Cdk) kinase families are known to regulate multiple events throughout cell division, whereas another kinase, citron kinase (CIT-K), for a long time has been considered to function solely during cytokinesis, the last phase of cell division. CIT-K was originally proposed to regulate the ingression of the cleavage furrow that forms at the equatorial cortex of the dividing cell after chromosome segregation. However, studies in the last decade have clarified that this kinase is, instead, required for the organization of the midbody in late cytokinesis, and also revealed novel functions of CIT-K earlier in mitosis and in DNA damage control. Moreover, CIT-K mutations have recently been linked to the development of human microcephaly, and CIT-K has been identified as a potential target in cancer therapy. In this Commentary, I describe and re-evaluate the functions and regulation of CIT-K during cell division and its involvement in human disease. Finally, I offer my perspectives on the open questions and future challenges that are necessary to address, in order to fully understand this important and yet unjustly neglected mitotic kinase. © 2017. Published by The Company of Biologists Ltd.

  16. WNT5A induces osteogenic differentiation of human adipose stem cells via rho-associated kinase Rock

    NARCIS (Netherlands)

    Santos, A.; Bakker, A.D.; de Blieck-Hogervorst, J.M.A.; Klein-Nulend, J.

    2010-01-01

    Background aims. Human (h) adipose tissue-derived mesenchymal stromal cells (ASC) constitute an interesting cellular source for bone tissue engineering applications. Wnts, for example Wnt5a, are probably important regulators of osteogenic differentiation of stem cells, but the role of Wnt5a in hASC

  17. Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates

    DEFF Research Database (Denmark)

    Hoffman, Nolan J; Parker, Benjamin L; Chaudhuri, Rima

    2015-01-01

    Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high...

  18. Effect of the cancer specific shorter form of human 6-phosphofructo-1-kinase on the metabolism of the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Andrejc, Darjan; Možir, Alenka; Legiša, Matic

    2017-05-08

    At first glance, there appears to be a high degree of similarity between the metabolism of yeast (the Crabtree effect) and human cancer cells (the Warburg effect). At the root of both effects is accelerated metabolic flow through glycolysis which leads to overflows of ethanol and lactic acid, respectively. It has been proposed that enhanced glycolytic flow in cancer cells is triggered by the altered kinetic characteristics of the key glycolytic regulatory enzyme 6-phosphofructo-1-kinase (Pfk). Through a posttranslational modification, highly active shorter Pfk-M fragments, which are resistant to feedback inhibition, are formed after the proteolytic cleavage of the C-terminus of the native human Pfk-M. Alternatively, enhanced glycolysis is triggered by optimal growth conditions in the yeast Saccharomyces cerevisiae. To assess the deregulation of glycolysis in yeast cells, the sfPFKM gene encoding highly active human shorter Pfk-M fragments was introduced into pfk-null S. cerevisiae. No growth of the transformants with the sfPFKM gene was observed on glucose and fructose. Glucose even induced rapid deactivation of Pfk1 activities in such transformants. However, Pfk1 activities of the sfPFKM transformants were detected in maltose medium, but the growth in maltose was possible only after the addition of 10 mM of ethanol to the medium. Ethanol seemed to alleviate the severely unbalanced NADH/NADPH ratio in the sfPFKM cells. However, the transformants carrying modified Pfk-M enzymes grew faster than the transformants with the human native human Pfk-M enzyme in a narrow ecological niche with a low maltose concentration medium that was further improved by additional modifications. Interestingly, periodic extracellular accumulation of phenylacetaldehyde was detected during the growth of the strain with modified Pfk-M but not with the strain encoding the human native enzyme. Highly active cancer-specific shorter Pfk-M fragments appear to trigger several controlling

  19. Equitoxic Doses of 5-Azacytidine and 5-Aza-2 ′ Deoxycytidine Induce Diverse Immediate and Overlapping Heritable Changes in the Transcriptome

    DEFF Research Database (Denmark)

    Qiu, X.; Hother, C.; Ralfkiær, U. M.

    2010-01-01

    BACKGROUND: The hypomethylating agent 5-Azacytidine (5-Aza-CR) is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS). Surprisingly, the deoxyribonucleoside analog 5-Aza-2'deoxycytidine (5-Aza-CdR) did not have a similar effect on survival in a large clinical...... line were treated with equitoxic doses of 5-Aza-CR and 5-Aza-CdR for 24 hrs, and the immediate (day 2) and lasting (day 8) effects on RNA expression examined. There was considerable overlap between the RNAs heritably upregulated by both drugs on day 8 but more RNAs were stably induced by the deoxy...... was observed in T24 cells. CONCLUSIONS/SIGNIFICANCE: Overall, this suggests that significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping....

  20. Human telomerase reverse-transcriptase promoter-controlled and herpes simplex virus thymidine kinase-armed adenoviruses for renal cell carcinoma treatment

    Directory of Open Access Journals (Sweden)

    Tian DW

    2013-04-01

    Full Text Available Dawei Tian,1–4 Yan Sun,3 Yang Yang,2,3 Mingde Lei,3 Na Ding,3 Ruifa Han2,31Tianjin Medical University, Tianjin, People's Republic of China; 2Department of Urinary Surgery, 3Tianjin Institute of Urology, Second Hospital of Tianjin Medical University, Tianjin, People's Republic of China; 4Tianjin Nankai Hospital, Tianjin, People's Republic of ChinaAbstract: New treatment strategies are required for renal cell carcinoma (RCC due to its relative insensitivity to conventional radio- and chemotherapies. The promising strategy of tumor inhibition using human telomerase reverse transcriptase (hTERT-controlled herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV in the hTERT promoter-driven HSV-TK/GCV suicide gene system was investigated. Tumor volume, weight, relative proliferation rate, and cell-apoptosis levels were examined in mice injected with adenovirus (Ad-hTERT-HSV-TK and GCV. Increased cell death occurred following treatment with Ads carrying hTERT-HSV-TK/GCV or cytomegalovirus promoter-controlled (CMV-HSV-TK/GCV for human RCC 786-0 and fibroblast MRC-5 cells. In mice, Ad-hTERT-HSV-TK/GCV more specifically inhibited tumor and RCC xenograft growth than Ad-CMV-HSV-TK/GCV (P < 0.05. Furthermore, Ad-hTERT-HSV-TK/GCV did not significantly damage normal fibroblasts or organ systems (heart, lung, liver, brain, kidney, and spleen. Thus, Ad-hTERT-HSV-TK/GCV is an effective RCC inhibitor in human cells in vitro and in vivo mouse models, indicating potential usefulness in RCC-targeted gene therapy.Keywords: hTERT promoter, HSV-TK/GCV, renal cell carcinoma, adenovirus

  1. Cryopreservation of Brain Endothelial Cells Derived from Human Induced Pluripotent Stem Cells Is Enhanced by Rho-Associated Coiled Coil-Containing Kinase Inhibition.

    Science.gov (United States)

    Wilson, Hannah K; Faubion, Madeline G; Hjortness, Michael K; Palecek, Sean P; Shusta, Eric V

    2016-12-01

    The blood-brain barrier (BBB) maintains brain homeostasis but also presents a major obstacle to brain drug delivery. Brain microvascular endothelial cells (BMECs) form the principal barrier and therefore represent the major cellular component of in vitro BBB models. Such models are often used for mechanistic studies of the BBB in health and disease and for drug screening. Recently, human induced pluripotent stem cells (iPSCs) have emerged as a new source for generating BMEC-like cells for use in in vitro human BBB studies. However, the inability to cryopreserve iPSC-BMECs has impeded implementation of this model by requiring a fresh differentiation to generate cells for each experiment. Cryopreservation of differentiated iPSC-BMECs would have a number of distinct advantages, including enabling production of larger scale lots, decreasing lead time to generate purified iPSC-BMEC cultures, and facilitating use of iPSC-BMECs in large-scale screening. In this study, we demonstrate that iPSC-BMECs can be successfully cryopreserved at multiple differentiation stages. Cryopreserved iPSC-BMECs retain high viability, express standard endothelial and BBB markers, and reach a high transendothelial electrical resistance (TEER) of ∼3000 Ω·cm2, equivalent to nonfrozen controls. Rho-associated coiled coil-containing kinase (ROCK) inhibitor Y-27632 substantially increased survival and attachment of cryopreserved iPSC-BMECs, as well as stabilized TEER above 800 Ω·cm2 out to 7 days post-thaw. Overall, cryopreservation will ease handling and storage of high-quality iPSC-BMECs, reducing a key barrier to greater implementation of these cells in modeling the human BBB.

  2. Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d.

    Science.gov (United States)

    Kero, Darko; Vukojevic, Katarina; Stazic, Petra; Sundov, Danijela; Mardesic Brakus, Snjezana; Saraga-Babic, Mirna

    2017-10-02

    Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19 INK4d . p19 INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19 INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19 INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19 INK4d throughout the investigated period indicates that p19 INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19 INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.

  3. Multiple roles of protein kinase a in arachidonic acid-mediated Ca2+ entry and tumor-derived human endothelial cell migration.

    Science.gov (United States)

    Fiorio Pla, Alessandra; Genova, Tullio; Pupo, Emanuela; Tomatis, Cristiana; Genazzani, Armando; Zaninetti, Roberta; Munaron, Luca

    2010-11-01

    We recently showed that arachidonic acid (AA) triggers calcium signals in endothelial cells derived from human breast carcinoma (B-TEC). In particular, AA-dependent Ca(2+) entry is involved in the early steps of tumor angiogenesis in vitro. Here, we investigated the multiple roles of the nitric oxide (NO) and cyclic AMP/protein kinase A (PKA) pathways in AA-mediated Ca(2+) signaling in the same cells. B-TEC stimulation with 5 μmol/L AA resulted in endothelial NO synthase (NOS) phosphorylation at Ser(1177), and NO release was measured with the fluorescent NO-sensitive probe DAR4M-AM. PKA inhibition by the use of the membrane-permeable PKA inhibitory peptide myristoylated PKI(14-22) completely prevented both AA- and NO-induced calcium entry and abolished B-TEC migration promoted by AA. AA-dependent calcium entry and cell migration were significantly affected by both the NOS inhibitor N(G)-nitro-l-arginine methyl ester and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, suggesting that NO release is functionally involved in the signaling dependent on AA. Moreover, pretreatment with carboxyamidotriazole, an antiangiogenic compound that interferes with agonist-activated calcium entry, prevented AA-dependent B-TEC motility. Interestingly, even in the absence of AA, enhancement of the cyclic AMP/PKA pathway with the adenylyl cyclase activator forskolin evoked a calcium entry dependent on NOS recruitment and NO release. The functional relevance of AA-induced calcium entry could be restricted to tumor-derived endothelial cells (EC) because AA evoked a smaller calcium entry in normal human microvascular ECs compared with B-TECs, and even more importantly, it was unable to promote cell motility in wound healing assay. This evidence opens an intriguing opportunity for differential pharmacologic treatment between normal and tumor-derived human ECs. ©2010 AACR.

  4. RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib.

    Science.gov (United States)

    Bradford, James R; Farren, Matthew; Powell, Steve J; Runswick, Sarah; Weston, Susie L; Brown, Helen; Delpuech, Oona; Wappett, Mark; Smith, Neil R; Carr, T Hedley; Dry, Jonathan R; Gibson, Neil J; Barry, Simon T

    2013-01-01

    Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers.

  5. A human osteosarcoma cell line expressing herpes simplex type-1 thymidine kinase: studies with radiolabeled (E)-5-(2-iodovinyl)-2'-fluoro-2'-deoxyuridine

    Energy Technology Data Exchange (ETDEWEB)

    Morin, Kevin W. [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada); Duan Weili [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada); Knaus, Edward E. [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada); McEwan, Alexander J.B. [Department of Radiology and Diagnostic Imaging, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, T6G 2N8 (Canada); Wiebe, Leonard I. [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, T6G 2N8 (Canada)]. E-mail: leonard.wiebe@ualberta.ca

    2005-07-01

    Introduction: (E)-5-(2-Iodovinyl)-2'-fluoro-2'-deoxyuridine (IVFRU) is a pyrimidine nucleoside analogue that accumulates selectively in murine cells expressing herpes simplex type-1 thymidine kinase (HSV-1 TK). The uptake of [{sup 125}I]IVFRU in human 143B osteosarcoma cells transduced with a retroviral vector bearing the HSV-1 TK gene (143B-LTK cells) is now reported. Methods: HSV-1 TK gene expression in 143B-LTK cells was confirmed by Western blotting and reverse transcriptase (RT)-PCR. Cell and subcellular uptake of [{sup 125}I]IVFRU was determined in cell culture, and whole body biodistribution after intravenous injection of [{sup 125}I]IVFRU was determined using nude mice bearing implanted 143B or 143B-LTK tumors. Results: Although IVFRU was less toxic to the human cell line expressing HSV-1 TK (143B-LTK) than ganciclovir, both IVFRU and ganciclovir were not toxic to the cell line not expressing HSV-1 TK (143B). When cells were exposed to [{sup 125}I]IVFRU in vitro, only the 143B-LTK cells accumulated radioactivity. The acid-soluble fraction from 143B-LTK cell lysates contained 8-fold greater activity than the acid-insoluble fraction after an 8-h exposure to [{sup 125}I]IVFRU. Biodistribution of [{sup 125}I]IVFRU in nude mice bearing subcutaneous 143B and 143B-LTK tumors revealed widespread distribution of the nucleoside in vivo but with specific localization in 143B-LTK tumors. Conclusion: The underlying biochemical process of metabolic entrapment of IVFRU in human osteosarcoma cells expressing HSV-1 TK is responsible for selective localization in these cells. The differences in subcellular distribution into the nucleic acid fraction, and in cytotoxicity, reflect the importance of cell type and lineage as determinants of the performance of gene imaging radiopharmaceuticals.

  6. Human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase.

    Science.gov (United States)

    Liu, Xiao; Pichulik, Tica; Wolz, Olaf-Oliver; Dang, Truong-Minh; Stutz, Andrea; Dillen, Carly; Delmiro Garcia, Magno; Kraus, Helene; Dickhöfer, Sabine; Daiber, Ellen; Münzenmayer, Lisa; Wahl, Silke; Rieber, Nikolaus; Kümmerle-Deschner, Jasmin; Yazdi, Amir; Franz-Wachtel, Mirita; Macek, Boris; Radsak, Markus; Vogel, Sebastian; Schulte, Berit; Walz, Juliane Sarah; Hartl, Dominik; Latz, Eicke; Stilgenbauer, Stephan; Grimbacher, Bodo; Miller, Lloyd; Brunner, Cornelia; Wolz, Christiane; Weber, Alexander N R

    2017-10-01

    The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1β processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1β release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1β processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK. Copyright © 2017 American Academy of Allergy

  7. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kuwasako, Kenji, E-mail: kuwasako@med.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Sekiguchi, Toshio [Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa, 927-0553 (Japan); Nagata, Sayaka [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Jiang, Danfeng; Hayashi, Hidetaka [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Murakami, Manabu [Department of Pharmacology, Hirosaki University, Graduate School of Medicine, Hirosaki, 036-8562 (Japan); Hattori, Yuichi [Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194 (Japan); Kitamura, Kazuo [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan)

    2016-02-19

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM{sub 1} receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM{sub 1} receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM{sub 1} receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [{sup 125}I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β{sub 2}-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM{sub 1} receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  8. RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib.

    Directory of Open Access Journals (Sweden)

    James R Bradford

    Full Text Available Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers.

  9. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Minyong Kang

    2017-02-01

    Full Text Available Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1, chloroquine (CQ and 3-methyladenine (3-MA were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8 assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating

  10. Raf kinase inhibitor protein (RKIP) inhibits the cell migration and invasion in human glioma cell lines in vitro.

    Science.gov (United States)

    Lei, Xuhui; Chang, Liang; Ye, Wei; Jiang, Chuanlu; Zhang, Zhiren

    2015-01-01

    To investigate the effects and the potential mechanisms of RKIP on cell migration, invasion and proliferation in human glioma cell lines in vitro. The RKIP over-expressing and RKIP knockdown human U87 glioma cells were used to reveal the effects of RKIP on human glioma cells migration, invasion and proliferation. After the recombinant plasmid pcDNA3.0-RKIP or RKIP-shRNA was transfected into the cell lines U87 by the means of liposome assay, the cells migration, invasion and proliferation were detected by wound healing, Transwell and MTT assay. Then, the levels of RKIP, MMP-3, MMP-9 and HMGA2 mRNA transcription were measured by means of RT-qPCR and levels of proteins expressions were determined using Western blot. The results of MTT assay suggested that the PKIP have little inhibitive effects on glioma cells proliferation (P>0.05). The present paper showed that the migration distances in the group of RKIP-shRNA were markedly increased compared to the pcDNA3.0-RKIP and control. Similarly, the results showed that the numbers of invasion cells in RKIP-shRNA were remarkably increased than the pcDNA3.0-RKIP group and control group. Western blot and RT-qPCR suggested that over-expressions of RKIP lessened the MMP-2, MMP-9 and HMGA2 expression, however, turning down the RKIP expression showed the inverse effects. RKIP inhibits the cells migrations and invasions. Meanwhile, RKIP might inhibit the glioma cells through inhibiting MMPs and HMAG2 expression. Therefore, we demonstrated that RKIP is an underlying target for the treatment of glioma.

  11. Systems Approach Reveals Nuclear Factor Erythroid 2-Related Factor 2/Protein Kinase R Crosstalk in Human Cutaneous Leishmaniasis.

    Science.gov (United States)

    Vivarini, Áislan de Carvalho; Calegari-Silva, Teresa Cristina; Saliba, Alessandra Mattos; Boaventura, Viviane Sampaio; França-Costa, Jaqueline; Khouri, Ricardo; Dierckx, Tim; Dias-Teixeira, Karina Luiza; Fasel, Nicolas; Barral, Aldina Maria Prado; Borges, Valéria Matos; Van Weyenbergh, Johan; Lopes, Ulisses Gazos

    2017-01-01

    Leishmania parasites infect macrophages, causing a wide spectrum of human diseases, from cutaneous to visceral forms. In search of novel therapeutic targets, we performed comprehensive in vitro and ex vivo mapping of the signaling pathways upstream and downstream of antioxidant transcription factor [nuclear factor erythroid 2-related factor 2 (Nrf2)] in cutaneous leishmaniasis (CL), by combining functional assays in human and murine macrophages with a systems biology analysis of in situ (skin biopsies) CL patient samples. First, we show the PKR pathway controls the expression and activation of Nrf2 in Leishmania amazonensis infection in vitro. Nrf2 activation also required PI3K/Akt signaling and autophagy mechanisms. Nrf2- or PKR/Akt-deficient macrophages exhibited increased levels of ROS/RNS and reduced expression of Sod1 Nrf2-dependent gene and reduced parasite load. L. amazonensis counteracted the Nrf2 inhibitor Keap1 through the upregulation of p62 via PKR. This Nrf2/Keap1 observation was confirmed in situ in skin biopsies from Leishmania-infected patients. Next, we explored the ex vivo transcriptome in CL patients, as compared to healthy controls. We found the antioxidant response element/Nrf2 signaling pathway was significantly upregulated in CL, including downstream target p62. In silico enrichment analysis confirmed upstream signaling by interferon and PI3K/Akt, and validated our in vitro findings. Our integrated in vitro, ex vivo, and in silico approach establish Nrf2 as a central player in human cutaneous leishmaniasis and reveal Nrf2/PKR crosstalk and PI3K/Akt pathways as potential therapeutic targets.

  12. Soluble Human Leukocyte Antigen-G5 Activates Extracellular Signal-Regulated Protein Kinase Signaling and Stimulates Trophoblast Invasion

    OpenAIRE

    YiFan Guo; Cheuk-Lun Lee; Kam-Hei So; Jing Gao; Yeung, William S. B.; YuanQing Yao; Kai-Fai Lee

    2013-01-01

    Soluble human leukocyte antigen-G (HLA-G) is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and ac...

  13. Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering.

    Science.gov (United States)

    Moyer, Tyler C; Holland, Andrew J

    2015-01-01

    The ability to rapidly and specifically modify the genome of mammalian cells has been a long-term goal of biomedical researchers. Recently, the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system from bacteria has been exploited for genome engineering in human cells. The CRISPR system directs the RNA-guided Cas9 nuclease to a specific genomic locus to induce a DNA double-strand break that may be subsequently repaired by homology-directed repair using an exogenous DNA repair template. Here we describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of a point mutation in human cells. Using this method, homozygous clonal cell lines can be constructed in 5-6 weeks. This method can also be adapted to insert larger DNA elements, such as fluorescent proteins and degrons, at defined genomic locations. CRISPR/Cas9 genome engineering offers exciting applications in both basic science and translational research. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Extracellular Signal-Regulated Kinase 5 is Required for Low-Concentration H2O2-Induced Angiogenesis of Human Umbilical Vein Endothelial Cells.

    Science.gov (United States)

    Jiang, Shan; Zhang, Dongxin; Huang, Hong; Lei, Yonghong; Han, Yan; Han, Weidong

    2017-01-01

    Background. The aim of this study was to assess the effects of low concentrations of H2O2 on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro and explore the underlying mechanisms. Methods. HUVECs were cultured and stimulated with different concentrations of H2O2. Flow cytometric analysis was used to select an optimal concentration of H2O2 for the following experiments. Cell proliferation, migration, and tubule formation were evaluated by Cell Counting Kit-8 (CCK-8) assays, scratch wound assays, and Matrigel tubule formation assays, respectively. For gain and loss of function studies, constitutively active MEK5 (CA-MEK5) and ERK5 shRNA lentiviruses were used to activate or knock down extracellular signal-regulated kinase 5 (ERK5). Results. We found that low concentrations of H2O2 promoted HUVECs proliferation, migration, and tubule formation. ERK5 in HUVECs was significantly activated by H2O2. Enhanced ERK5 activity significantly amplified the proangiogenic effects of H2O2; in contrast, ERK5 knock-down abrogated the effects of H2O2. Conclusions. Our results confirmed that low concentrations of H2O2 promoted HUVECs angiogenesis in vitro, and ERK5 is an essential mediator of this process. Therefore, ERK5 may be a potential therapeutic target for promoting angiogenesis and improving graft survival.

  15. Body weight management effect of burdock (Arctium lappa L.) root is associated with the activation of AMP-activated protein kinase in human HepG2 cells.

    Science.gov (United States)

    Kuo, Daih-Huang; Hung, Ming-Chi; Hung, Chao-Ming; Liu, Li-Min; Chen, Fu-An; Shieh, Po-Chuen; Ho, Chi-Tang; Way, Tzong-Der

    2012-10-01

    Burdock (Arcticum lappa L.) root is used in folk medicine and also as a vegetable in Asian countries. In the present study, burdock root treatment significantly reduced body weight in rats. To evaluate the bioactive compounds, we successively extracted the burdock root with ethanol (AL-1), and fractionated it with n-hexane (AL-2), ethyl acetate (AL-3), n-butanol (AL-4), and water (AL-5). Among these fractions, AL-2 contained components with the most effective hypolipidemic potential in human hepatoma HepG2 cells. AL-2 decreased the expression of fatty acid synthase (FASN) and inhibited the activity of acetyl-coenzyme A carboxylase (ACC) by stimulating AMP-activated protein kinase (AMPK) through the LKB1 pathway. Three active compounds were identified from the AL-2, namely α-linolenic acid, methyl α-linolenate, and methyl oleate. These results suggest that burdock root is expected to be useful for body weight management. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Cell culture condition-dependent impact of AGE-rich food extracts on kinase activation and cell survival on human fibroblasts.

    Science.gov (United States)

    Nass, Norbert; Weissenberg, Kristian; Somoza, Veronika; Ruhs, Stefanie; Silber, Rolf-Edgar; Simm, Andreas

    2014-03-01

    Advanced glycation end products (AGEs) are stable end products of the Maillard reaction. Effects of food extracts are often initially analysed in cellular test systems and it is not clear how different cell culture conditions might influence the results. Therefore, we compared the effects of two models for AGE-rich food, bread crust and coffee extract (CE) on WI-38 human lung fibroblasts under different cell culture conditions (sub-confluent versus confluent cells, with and without serum). WI-38 cells responded to coffee and bread crust extract (BCE) with a rapid phosphorylation of PKB (AKT), p42/44 MAPK (ERK 1/2) and p38 MAPK, strongly depending on culture conditions. BCE resulted in increased cell numbers, whereas CE appeared to be cytotoxic. When cell numbers under all culture conditions and treatments were correlated with kinase phosphorylation, the relation between phospho-p38 MAPK and phospho-AKT represented a good, cell culture condition-independent predictor of cell survival.

  17. ITC-derived binding affinity may be biased due to titrant (nano-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2.

    Directory of Open Access Journals (Sweden)

    Maria Winiewska

    Full Text Available The binding of four bromobenzotriazoles to the catalytic subunit of human protein kinase CK2 was assessed by two complementary methods: Microscale Thermophoresis (MST and Isothermal Titration Calorimetry (ITC. New algorithm proposed for the global analysis of MST pseudo-titration data enabled reliable determination of binding affinities for two distinct sites, a relatively strong one with the Kd of the order of 100 nM and a substantially weaker one (Kd > 1 μM. The affinities for the strong binding site determined for the same protein-ligand systems using ITC were in most cases approximately 10-fold underestimated. The discrepancy was assigned directly to the kinetics of ligand nano-aggregates decay occurring upon injection of the concentrated ligand solution to the protein sample. The binding affinities determined in the reverse ITC experiment, in which ligands were titrated with a concentrated protein solution, agreed with the MST-derived data. Our analysis suggests that some ITC-derived Kd values, routinely reported together with PDB structures of protein-ligand complexes, may be biased due to the uncontrolled ligand (nano-aggregation, which may occur even substantially below the solubility limit.

  18. ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2

    Science.gov (United States)

    Winiewska, Maria; Bugajska, Ewa

    2017-01-01

    The binding of four bromobenzotriazoles to the catalytic subunit of human protein kinase CK2 was assessed by two complementary methods: Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC). New algorithm proposed for the global analysis of MST pseudo-titration data enabled reliable determination of binding affinities for two distinct sites, a relatively strong one with the Kd of the order of 100 nM and a substantially weaker one (Kd > 1 μM). The affinities for the strong binding site determined for the same protein-ligand systems using ITC were in most cases approximately 10-fold underestimated. The discrepancy was assigned directly to the kinetics of ligand nano-aggregates decay occurring upon injection of the concentrated ligand solution to the protein sample. The binding affinities determined in the reverse ITC experiment, in which ligands were titrated with a concentrated protein solution, agreed with the MST-derived data. Our analysis suggests that some ITC-derived Kd values, routinely reported together with PDB structures of protein-ligand complexes, may be biased due to the uncontrolled ligand (nano)-aggregation, which may occur even substantially below the solubility limit. PMID:28273138

  19. Apoptosis-related molecular differences for response to tyrosin kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells

    Directory of Open Access Journals (Sweden)

    Jixia Li

    2013-01-01

    Full Text Available Context: The epidermal growth factor receptor (EGFR family is reportedly overexpressed in bladder cancer, and tyrosine kinaseinhibitors (TKIs have been suggested as treatment. Gefitinib is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and HER2 (human EGFR type 2 receptor. Both compounds compete with the binding of adenosine triphosphate (ATP to the tyrosine kinase domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a major clinical problem. Aims: To compare the apoptosis signaling pathway(s induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive and UM-UC-14 (drug-resistant bladder cancer cells and to identify molecular differences that might be useful predictors of their efficacy. Materials and Methods: Cell proliferation, cell cycle and apoptosis assay were used to detect the effect of TKIs on UM-UC-5 and UM-UC-14 cells. Molecular differences for response to TKIs were examined by protein array. Results: TKIs strongly inhibited cell proliferation and induced cell cycle G1 arrest and apoptosis in UM-UC-5 cells. Most notable apoptosis molecular differences included decreased claspin, trail, and survivin by TKIs in the sensitive cells. In contrast, TKIs had no effect on resistant cells. Conclusions: Claspin, trail, and survivin might be used to determine the sensitivity of bladder cancers to TKIs.

  20. Phosphatidylinositol 3-kinases are involved in the all-trans retinoic acid-induced upregulation of CD38 antigen on human haematopoietic cells.

    Science.gov (United States)

    Lewandowski, Daniel; Linassier, Claude; Iochmann, Sophie; Degenne, Michel; Domenech, Jorge; Colombat, Philippe; Binet, Christian; Hérault, Olivier

    2002-08-01

    All-trans retinoic acid (ATRA) is a specific inducer of CD38 antigen on marrow CD34+ cells as well as on blast cells in acute promyelocytic and myeloblastic leukaemia. The CD38 antigen contributes to the control of blast cell proliferation, and the upregulation of CD38 might constitute an element in the pathogenesis of retinoic acid syndrome. The aim of this study was to determine whether phosphoinositide 3-kinase (PI3-K) is involved in the modification of CD38 antigen expression on myeloid cells, as PI3-K plays a major role in the ATRA-induced granulocytic differentiation of HL-60 cells. We evaluated the effects of PI3-K inhibitors (wortmannin and LY294002) on the levels of CD38 antigen and mRNA in HL-60 and normal marrow CD34+ cells exposed to ATRA (1 micromol/l). The inhibitors prevented increase in CD38 mRNA expression and the overexpression of membrane CD38 antigen, without modification of the cytoplasmic level of this antigen. Interestingly, PI3-K activity was also necessary for CD38 expression on normal marrow CD34+ cells and for the ATRA-induced upregulation of CD157, a CD38-related antigen. In conclusion, PI3-K activity plays an essential role in the regulation of CD38 expression on human haematopoietic cells, and might constitute an interesting therapeutic target in haematological disorders involving CD38 overexpression.

  1. Saw palmetto extract suppresses insulin-like growth factor-I signaling and induces stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation in human prostate epithelial cells.

    Science.gov (United States)

    Wadsworth, Teri L; Carroll, Julie M; Mallinson, Rebecca A; Roberts, Charles T; Roselli, Charles E

    2004-07-01

    A common alternative therapy for benign prostatic hyperplasia (BPH) is the extract from the fruit of saw palmetto (SPE). BPH is caused by nonmalignant growth of epithelial and stromal elements of the prostate. IGF action is important for prostate growth and development, and changes in the IGF system have been documented in BPH tissues. The main signaling pathways activated by the binding of IGF-I to the IGF-I receptor (IGF-IR) are the ERK arm of the MAPK cascade and the phosphoinositol-3-kinase (PI3K)/protein kinase B (PKB/Akt) cascade. We tested the hypothesis that SPE suppresses growth and induces apoptosis in the P69 prostate epithelial cell line by inhibiting IGF-I signaling. Treatment with 150 microg/ml SPE for 24 h decreased IGF-I-induced proliferation of P69 cells and induced cleavage of the enzyme poly(ADP-ribose)polymerase (PARP), an index of apoptosis. Treatment of serum-starved P69 cells with 150 microg/ml SPE for 6 h reduced IGF-I-induced phosphorylation of Akt (assessed by Western blot) and Akt activity (assessed by an Akt kinase assay). Western blot analysis showed that SPE reduced IGF-I-induced phosphorylation of the adapter protein insulin receptor substrate-1 and decreased downstream effects of Akt activation, including increased cyclin D1 levels and phosphorylation of glycogen synthase kinase-3 and p70(s6k). There was no effect on IGF-I-induced phosphorylation of MAPK, IGF-IR, or Shc. Treatment of starved cells with SPE alone induced phosphorylation the proapoptotic protein JNK. SPE treatment may relieve symptoms of BPH, in part, by inhibiting specific components of the IGF-I signaling pathway and inducing JNK activation, thus mediating antiproliferative and proapoptotic effects on prostate epithelia.

  2. Side-effects of protein kinase inhibitors on ion channels

    Indian Academy of Sciences (India)

    Protein kinases are one of the largest gene families and have regulatory roles in all aspects of eukaryotic cell function. Modulation of protein kinase activity is a desirable therapeutic approach for a number of human diseases associated with aberrant kinase activity, including cancers, arthritis and cardiovascular disorders.

  3. Basic fibroblast growth factor induces matrix metalloproteinase-13 via ERK MAP kinase-altered phosphorylation and sumoylation of Elk-1 in human adult articular chondrocytes

    Directory of Open Access Journals (Sweden)

    Hee-Jeong Im

    2009-10-01

    Full Text Available Hee-Jeong Im,1–4 Andrew D Sharrocks,5 Xia Lin,6 Dongyao Yan,1 Jaesung Kim,1 Andre J van Wijnen,7 Robert A Hipskind81Departments of Biochemistry, 2Internal Medicine, 3Section of Rheumatology, Orthopedic Surgery, 4Rush University Medical Center, and Department of Bioengineering; University of Illinois at Chicago, IL USA; 5Faculty of Life Sciences, University of Manchester, Oxford Rd, Manchester, UK; 6Michael D DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, USA; 7Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA; 8Institute De Genetique Moleculaire de Montpellier, FranceAbstract: Degradation of the extracellular matrix (ECM by matrix metalloproteinases (MMPs and release of basic fibroblast growth factor (bFGF are principal aspects of the pathology of osteoarthritis (OA. ECM disruption leads to bFGF release, which activates the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK pathway and its downstream target the Ets-like transcription factor Elk-1. Previously we demonstrated that the bFGF-ERK-Elk-1 signaling axis is responsible for the potent induction of MMP-13 in human primary articular chondrocytes. Here we report that, in addition to phosphorylation of Elk-1, dynamic posttranslational modification of Elk-1 by small ubiquitin-related modifier (SUMO serves as an important mechanism through which MMP-13 gene expression is regulated. We show that bFGF activates Elk-1 mainly through the ERK pathway and that increased phosphorylation of Elk-1 is accompanied by decreased conjugation of SUMO to Elk-1. Reporter gene assays reveal that phosphorylation renders Elk-1 competent for induction of MMP-13 gene transcription, while sumoylation has the opposite effect. Furthermore, we demonstrate that the SUMO-conjugase Ubc9 acts as a key mediator for Elk-1 sumoylation. Taken together, our results suggest that sumoylation antagonizes the phosphorylation

  4. Gene expression patterns that predict sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors in lung cancer cell lines and human lung tumors

    Directory of Open Access Journals (Sweden)

    Haura Eric B

    2006-11-01

    Full Text Available Abstract Background Increased focus surrounds identifying patients with advanced non-small cell lung cancer (NSCLC who will benefit from treatment with epidermal growth factor receptor (EGFR tyrosine kinase inhibitors (TKI. EGFR mutation, gene copy number, coexpression of ErbB proteins and ligands, and epithelial to mesenchymal transition markers all correlate with EGFR TKI sensitivity, and while prediction of sensitivity using any one of the markers does identify responders, individual markers do not encompass all potential responders due to high levels of inter-patient and inter-tumor variability. We hypothesized that a multivariate predictor of EGFR TKI sensitivity based on gene expression data would offer a clinically useful method of accounting for the increased variability inherent in predicting response to EGFR TKI and for elucidation of mechanisms of aberrant EGFR signalling. Furthermore, we anticipated that this methodology would result in improved predictions compared to single parameters alone both in vitro and in vivo. Results Gene expression data derived from cell lines that demonstrate differential sensitivity to EGFR TKI, such as erlotinib, were used to generate models for a priori prediction of response. The gene expression signature of EGFR TKI sensitivity displays significant biological relevance in lung cancer biology in that pertinent signalling molecules and downstream effector molecules are present in the signature. Diagonal linear discriminant analysis using this gene signature was highly effective in classifying out-of-sample cancer cell lines by sensitivity to EGFR inhibition, and was more accurate than classifying by mutational status alone. Using the same predictor, we classified human lung adenocarcinomas and captured the majority of tumors with high levels of EGFR activation as well as those harbouring activating mutations in the kinase domain. We have demonstrated that predictive models of EGFR TKI sensitivity can

  5. A novel Physarum polycephalum SR protein kinase specifically phosphorylates the RS domain of the human SR protein, ASF/SF2.

    Science.gov (United States)

    Liu, Shide; Kang, Kang; Zhang, Jianhua; Ouyang, Qiuling; Zhou, Zhuolong; Tian, Shengli; Xing, Miao

    2009-08-01

    A 1591-bp cDNA of a serine-rich protein kinase (SRPK)-like protein has been identified in Physarum polycephalum (GenBank accession No. DQ140379). The cDNA contains two repeat sequences at bp 1-153 and bp 395-547. The encoding sequence is 56% homologous to human SRPK1 and is named Physarum SRPK (PSRPK). Consistent with other SRPKs, the consensus motifs of PSRPK are within the two conserved domains (CDs). However, divergent motifs between the N-terminal and CDs are much shorter than the corresponding sequences of other SRPKs. To study the structure and function of this protein, we performed co-expression experiment in Escherichia coli and in vitro phosphorylation assay to investigate the phosphorylation effect of recombinant PSRPK on the human SR protein, ASF/SF2. Western blot analysis showed that PSRPK could phosphorylate ASF/SF2 in E. coli cells. Autoradiographic examination showed that both recombinant PSRPK and a truncated form of PSRPK with a 28-aa deletion at the N-terminus could phosphorylate ASF/SF2 and a truncated form of ASF/SF2 that contains the RS domain. However, these two forms of PSRPK could not phosphorylate a truncated form ASF/SF2 that lacks the RS domain. A truncated form of PSRPK that lacks either of CDs does not have any phosphorylation activity. These results indicated that, like other SRPKs, the phosphorylation site in PSRPK is located within the RS domain of the SR protein and that its phosphorylation activity is closely associated with the two CDs. This study on the structure and function of PSRPK demonstrates that it is a new member of the SRPK family.

  6. Fasting Induces Nuclear Factor E2-Related Factor 2 and ATP-Binding Cassette Transporters via Protein Kinase A and Sirtuin-1 in Mouse and Human

    Science.gov (United States)

    Kulkarni, Supriya R.; Donepudi, Ajay C.; Xu, Jialin; Wei, Wei; Cheng, Qiuqiong C.; Driscoll, Maureen V.; Johnson, Delinda A.; Johnson, Jeffrey A.; Li, Xiaoling

    2014-01-01

    Abstract Aims: The purpose of this study was to determine whether 3′-5′-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2–4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. Results: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1–dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. Innovation: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. Conclusion: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway. Antioxid. Redox Signal. 20, 15–30. PMID:23725046

  7. Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction.

    Science.gov (United States)

    Tugaeva, Kristina V; Tsvetkov, Philipp O; Sluchanko, Nikolai N

    2017-01-01

    Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3/phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Among many known phosphorylation sites within Tau, protein kinase A (PKA) phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau (pTau) are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3/pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3/pTau interaction, our co-expression system may be useful to study of other selected 14-3-3/phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods.

  8. Stable expression of human muscle-specific kinase in HEp-2 M4 cells for automatic immunofluorescence diagnostics of myasthenia gravis.

    Science.gov (United States)

    George, Sandra; Paulick, Silvia; Knütter, Ilka; Röber, Nadja; Hiemann, Rico; Roggenbuck, Dirk; Conrad, Karsten; Küpper, Jan-Heiner

    2014-01-01

    Muscle-specific kinase (MuSK) belongs to the nicotinic acetylcholine receptor complex which is targeted by pathogenic autoantibodies causing Myasthenia gravis. While up to 95% of patients with generalized Myasthenia gravis were shown to be positive for acetylcholine receptor-specific autoantibodies, up to 70% of the remaining patients develop autoantibodies against MuSK. Discrimination of the autoantibody specificity is important for therapy of Myasthenia gravis. Recently, the new automatic fluorescence assessment platform AKLIDES has been developed for immunofluorescence-based diagnostics of autoimmune diseases. In order to establish an AKLIDES procedure for the detection of MuSK-specific autoantibodies (anti-MuSK), we developed a recombinant HEp-2 cell clone expressing the human MuSK cDNA. Here we show at the mRNA and protein level that the cell clone HEp-2 M4 stably expresses human MuSK. We provide evidence for a localization of MuSK at the cell membrane. Using cell clone HEp-2 M4 on the AKLIDES system, we investigated 34 patient sera that were previously tested anti-MuSK positive by radioimmunoassay as positive controls. As negative controls, we tested 29 acetylcholine receptor-positive but MuSK-negative patient sera, 30 amytrophic lateral sclerosis (ALS) patient sera and 45 blood donors. HEp-2 M4 cells revealed a high specificity for the detection of MuSK autoantibodies from 25 patient sera assessed by a specific pattern on HEp-2 M4 cells. By using appropriate cell culture additives, the fraction of cells stained positive with anti-MuSK containing sera can be increased from 2-16% to 10-48%, depending on the serum. In conclusion, we provide data showing that the novel recombinant cell line HEp-2 M4 can be used to screen for anti-MuSK with the automatic AKLIDES system.

  9. Stable expression of human muscle-specific kinase in HEp-2 M4 cells for automatic immunofluorescence diagnostics of myasthenia gravis.

    Directory of Open Access Journals (Sweden)

    Sandra George

    Full Text Available Muscle-specific kinase (MuSK belongs to the nicotinic acetylcholine receptor complex which is targeted by pathogenic autoantibodies causing Myasthenia gravis. While up to 95% of patients with generalized Myasthenia gravis were shown to be positive for acetylcholine receptor-specific autoantibodies, up to 70% of the remaining patients develop autoantibodies against MuSK. Discrimination of the autoantibody specificity is important for therapy of Myasthenia gravis. Recently, the new automatic fluorescence assessment platform AKLIDES has been developed for immunofluorescence-based diagnostics of autoimmune diseases. In order to establish an AKLIDES procedure for the detection of MuSK-specific autoantibodies (anti-MuSK, we developed a recombinant HEp-2 cell clone expressing the human MuSK cDNA. Here we show at the mRNA and protein level that the cell clone HEp-2 M4 stably expresses human MuSK. We provide evidence for a localization of MuSK at the cell membrane. Using cell clone HEp-2 M4 on the AKLIDES system, we investigated 34 patient sera that were previously tested anti-MuSK positive by radioimmunoassay as positive controls. As negative controls, we tested 29 acetylcholine receptor-positive but MuSK-negative patient sera, 30 amytrophic lateral sclerosis (ALS patient sera and 45 blood donors. HEp-2 M4 cells revealed a high specificity for the detection of MuSK autoantibodies from 25 patient sera assessed by a specific pattern on HEp-2 M4 cells. By using appropriate cell culture additives, the fraction of cells stained positive with anti-MuSK containing sera can be increased from 2-16% to 10-48%, depending on the serum. In conclusion, we provide data showing that the novel recombinant cell line HEp-2 M4 can be used to screen for anti-MuSK with the automatic AKLIDES system.

  10. Expression of the vitamin K-dependent proteins GAS6 and protein S and the TAM receptor tyrosine kinases in human atherosclerotic carotid plaques.

    Science.gov (United States)

    Hurtado, B; Muñoz, X; Recarte-Pelz, P; García, N; Luque, A; Krupinski, J; Sala, N; García de Frutos, P

    2011-05-01

    The GAS6/ProS-TAM system is composed of two vitamin K-dependent ligands (GAS6 and protein S) and their three protein tyrosine kinase receptors TYRO3, AXL and MERTK, known as the TAM receptors. The system plays a prominent role in conditions of injury, inflammation and repair. In murine models of atherosclerotic plaque formation, mutations in its components affect atherosclerosis severity. Here we used Taqman low-density arrays and immunoblotting to study mRNA and protein expression of GAS6, ProS and the TAM receptors in human carotid arteries with different degrees of atherosclerosis. The results show a clear down-regulation of the expression of AXL in atheroma plaques with respect to normal carotids that is matched by decreased abundance of AXL in protein extracts detected by immunoblotting. A similar decrease was observed in PROS1 mRNA expression in atherosclerotic carotids compared to the normal ones, but in this case protein S (ProS) was clearly increased in protein extracts of carotid arteries with increasing grade of atherosclerosis, suggesting that ProS is carried into the plaque. MERTK was also increased in atherosclerotic carotid arteries with respect to the normal ones, suggesting that the ProS-MERTK axis is functional in advanced human atherosclerotic plaques. MERTK was expressed in macrophages, frequently in association with ProS, while ProS was abundant also in the necrotic core. Our data suggest that the ProS-MERTK ligand-receptor pair was active in advanced stages of atherosclerosis, while AXL signalling is probably down-regulated.

  11. Celecoxib treatment of fibrous dysplasia (FD) in a human FD cell line and FD-like lesions in mice with protein kinase A (PKA) defects.

    Science.gov (United States)

    Saloustros, Emmanouil; Liu, Sisi; Mertz, Edward L; Bhattacharyya, Nisan; Starost, Matthew F; Salpea, Paraskevi; Nesterova, Maria; Collins, Michael; Leikin, Sergey; Stratakis, Constantine A

    2017-01-05

    Osteochondromyxomas (OMX) in the context of Carney complex (CNC) and fibrous dysplasia (FD)-like lesions (FDLL) in mice, as well as isolated myxomas in humans may be caused by inactivation of PRKAR1A, the gene coding for the type 1a regulatory subunit (R1α) of cAMP-dependent protein kinase (PKA). OMXs and FDLL in mice lacking Prkar1a grow from abnormal proliferation of adult bone stromal cells (aBSCs). Prkar1a and Prkaca (coding for Cα) haploinsufficiency leads to COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of aBSCs. Celecoxib is a cyclooxygenase-2 (COX2) inhibitor. We hypothesized that COX-2 inhibition may have an effect in FD and FDLL. In vitro treatment of a human cell line prepared from a FD patient with Celecoxib resulted in decreased PGE2 and cell proliferation. Treatment of mice haploinsufficient for R1α and Cα with 1500 mg/kg Celecoxib led to decreased PGE2 and proliferation and increased apoptosis, with a corresponding gene expression profile, resulting in dramatic reduction of tumor growth. Furthermore, the treatment improved the organization of cortical bone that was adjacent to the tumor. We conclude that, in vitro and in vivo, Celecoxib had an inhibitory effect on FD cell proliferation and in mouse FDLL structure, respectively. We speculate that COX-2 inhibitors offer an attractive alternative to current treatments for benign tumors such as OMX and FD that, apart from tumor suppression, may mechanically stabilize affected bones. Published by Elsevier Ireland Ltd.

  12. Sphingosine kinase-1, S1P transporter spinster homolog 2 and S1P2 mRNA expressions are increased in liver with advanced fibrosis in human.

    Science.gov (United States)

    Sato, Masaya; Ikeda, Hitoshi; Uranbileg, Baasanjav; Kurano, Makoto; Saigusa, Daisuke; Aoki, Junken; Maki, Harufumi; Kudo, Hiroki; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka

    2016-08-26

    The role of sphingosine 1-phosphate (S1P) in liver fibrosis or inflammation was not fully examined in human. Controversy exists which S1P receptors, S1P1 and S1P3 vs S1P2, would be importantly involved in its mechanism. To clarify these matters, 80 patients who received liver resection for hepatocellular carcinoma and 9 patients for metastatic liver tumor were enrolled. S1P metabolism was analyzed in background, non-tumorous liver tissue. mRNA levels of sphingosine kinase 1 (SK1) but not SK2 were increased in livers with fibrosis stages 3-4 compared to those with 0-2 and to normal liver. However, S1P was not increased in advanced fibrotic liver, where mRNA levels of S1P transporter spinster homolog 2 (SPNS2) but not S1P-degrading enzymes were enhanced. Furthermore, mRNA levels of S1P2 but not S1P1 or S1P3 were increased in advanced fibrotic liver. These increased mRNA levels of SK1, SPNS2 and S1P2 in fibrotic liver were correlated with α-smooth muscle actin mRNA levels in liver, and with serum ALT levels. In conclusion, S1P may be actively generated, transported to outside the cells, and bind to its specific receptor in human liver to play a role in fibrosis or inflammation. Altered S1P metabolism in fibrotic liver may be their therapeutic target.

  13. Hyperphosphatemia induces cellular senescence in human aorta smooth muscle cells through integrin linked kinase (ILK) up-regulation.

    Science.gov (United States)

    Troyano, Nuria; Nogal, María Del; Mora, Inés; Diaz-Naves, Manuel; Lopez-Carrillo, Natalia; Sosa, Patricia; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruiz-Torres, María P

    2015-12-01

    Aging is conditioned by genetic and environmental factors. Hyperphosphatemia is related to some pathologies, affecting to vascular cells behavior. This work analyze whether high concentration of extracellular phosphate induces vascular smooth muscle cells senescence, exploring the intracellular mechanisms and highlighting the in vivo relevance of this phenomenon. Human aortic smooth muscle cells treated with β-Glycerophosphate (BGP, 10mM) suffered cellular senescence by increasing p53, p21 and p16 expression and the senescence associated β-galactosidase activity. In parallel, BGP induced ILK overexpression, dependent on the IGF-1 receptor activation, and oxidative stress. Down-regulating ILK expression prevented BGP-induced senescence and oxidative stress. Aortic rings from young rats treated with 10mM BGP for 48h, showed increased p53, p16 and ILK expression and SA-β-gal activity. Seven/eight nephrectomized rats feeding a hyperphosphatemic diet and fifteenth- month old mice showed hyperphosphatemia and aortic ILK, p53 and p16 expression. In conclusion, we demonstrated that high extracellular concentration of phosphate induced senescence in cultured smooth muscle through the activation of IGF-1 receptor and ILK overexpression and provided solid evidences for the in vivo relevance of these results since aged animals showed high levels of serum phosphate linked to increased expression of ILK and senescence genes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  14. CLP induces apoptosis in human leukemia K562 cells through Ca(2+) regulating extracellular-related protein kinase ERK activation.

    Science.gov (United States)

    Wang, C L; Ng, T B; Cao, X H; Jiang, Y; Liu, Z K; Wen, T Y; Liu, F

    2009-04-18

    The cyclic lipopeptide (CLP) has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in CLP-induced apoptosis are still uncharacterized in human leukemic K562 cells. The current study investigated the molecular mechanism of action of CLP, purified from Bacillus natto T-2. CLP-induced a sustained increase in concentration of intracellular Ca(2+). This increase in [Ca(2+)]i was associated with CLP-induced cell apoptosis and ERK phosphorylation. CLP-induced cell apoptosis was reversed by PD98059 (an inhibitor of ERK), but not by SB203580 (an inhibitor of p38) and SP200125 (an inhibitor of JNK), suggesting that the action of CLP on K562 cells was via ERK, but not via p38 and JNK. On the other hand, pretreatment with Bapta-AM, a well-known calcium chelator, partially blocked CLP-induced apoptosis, indicating that the elevation of [Ca(2+)]i may play an important role in the apoptosis. Collectively, in K562 cells, CLP-induced an increase in [Ca(2+)]i which evoked ERK phosphorylation. This ERK phosphorylation subsequently activated Bax, cytochrome c and caspase-3 leading to apoptosis.

  15. Proteomics reveal energy metabolism and mitogen-activated protein kinase signal transduction perturbation in human Borna disease virus Hu-H1-infected oligodendroglial cells.

    Science.gov (United States)

    Liu, X; Yang, Y; Zhao, M; Bode, L; Zhang, L; Pan, J; Lv, L; Zhan, Y; Liu, S; Zhang, L; Wang, X; Huang, R; Zhou, J; Xie, P

    2014-05-30

    Borna disease virus (BDV) is a neurotropic, non-cytolytic RNA virus which replicates in the cell nucleus targeting mainly hippocampal neurons, but also astroglial and oligodendroglial cells in the brain. BDV is associated with a large spectrum of neuropsychiatric pathologies in animals. Its relationship to human neuropsychiatric illness still remains controversial. We could recently demonstrate that human BDV strain Hu-H1 promoted apoptosis and inhibited cell proliferation in a human oligodendroglial cell line (OL cells) whereas laboratory BDV strain V acted contrariwise. Here, differential protein expression between BDV Hu-H1-infected OL cells and non-infected OL cells was assessed through a proteomics approach, using two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry. A total of 63 differential host proteins were identified in BDV Hu-H1-infected OL cells compared to non-infected OL cells. We found that most changes referred to alterations related to the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, the tricarboxylic acid (TCA) cycle, and glycolysis /gluconeogenesis. By manual querying, two differential proteins were found to be associated with mitogen-activated protein kinase (MAPK) signal transduction. Five key signaling proteins of this pathway (i.e., p-Raf, p-MEK, p-ERK1/2, p-RSK, and p-MSK) were selected for Western blotting validation. p-ERK1/2 and p-RSK were found to be significantly up-regulated, and p-MSK was found to be significantly down-regulated in BDV Hu-H1-infected OL cells compared to non-infected OL cell. Although BDV Hu-H1 constitutively activated the ERK-RSK pathway, host cell proliferation and nuclear translocation of activated pERK in BDV Hu-H1-infected OL cells were impaired. These findings indicate that BDV Hu-H1 infection of human oligodendroglial cells significantly perturbs host energy metabolism, activates the downstream ERK-RSK complex of

  16. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    Energy Technology Data Exchange (ETDEWEB)

    Gilmour, Peter S., E-mail: Peter.Gilmour@astrazeneca.com [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); O' Shea, Patrick J.; Fagura, Malbinder [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Pilling, James E. [Discovery Sciences, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Sanganee, Hitesh [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Wada, Hiroki [R and I IMed, AstraZeneca R and D, Molndal (Sweden); Courtney, Paul F. [DMPK, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Kavanagh, Stefan; Hall, Peter A. [Safety Assessment, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Escott, K. Jane [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom)

    2013-10-15

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis

  17. Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase

    DEFF Research Database (Denmark)

    Green, Charlotte Jane; Pedersen, Maria; Pedersen, Bente K

    2011-01-01

    To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes....

  18. A validated assay for the simultaneous quantification of six tyrosine kinase inhibitors and two active metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry

    NARCIS (Netherlands)

    Erp, N. van; Wit, D. de; Guchelaar, H.J.; Gelderblom, H.; Hessing, T.J.; Hartigh, J. den

    2013-01-01

    A sensitive, sophisticated and practical bioanalytical assay for the simultaneous determination of six tyrosine kinase inhibitors (imatinib, sunitinib, nilotinib, dasatinib, pazopanib, regorafenib) and two active metabolites (N-desmethyl imatinib and N-desethyl sunitinib) was developed and

  19. [Modulation by some fatty acids of protein kinase C-dependent NADPH oxidase in human adherent monocyte: mechanism of action, possible implication in atherogenesis].

    Science.gov (United States)

    Léger, C L; Kadri-Hassani, N

    1995-01-01

    It is largely admitted nowadays that the early stage of the atherosclerotic lesion involves formation of oxidized (and minimally oxidized) low-density lipoprotein. Their properties are briefly reviewed. It is recalled that a lipolytic process also takes place both at the lumenal surface and in the subendothelial space of the vessels implying lipoprotein lipase (LpL) activity. Recent studies emphasize the role of LpL in accumulating LDL in the vascular tissue (Rutledge & Golberg, J. Lipid Res., 1994, 35, 1152-1160), but the role of LpL-generated unesterified fatty acids (UEFA) in these two locations and their possible implication in atherogenesis are largely neglected. Physiological and pathophysiological significance of UEFA in the human adherent monocyte modulation of the superoxide anion (O2.-) production has been examined by our group, leading to a possible mechanism of modulation of LDL oxidative modification. The O2.- production-modulating effect of a 30-min UEFA preincubation has been studied in intact human adherent monocytes (HAM) after stimulation by a direct effector of protein kinase C (PKC). It has been established that UEFA alone (in the absence of PKC effectors) were not able to modulate the O2.- production of HAM whereas they had such a capacity in the presence of PKC effectors, phorbol myristate acetate (PMA) or diacylglycerol (DAG). In this case inhibitors of PKC such as GF 109203 X suppressed the modulating effect. UEFA have also been shown to possess a bimodal action in the presence of PKC effectors: they depressed or enhanced O2.- production at micromolar or nanomolar concentrations, respectively. All these results contrasted with others obtained in neutrophils or nonadherent monocytes, suggesting an absolute requirement of PKC for the phagocyte-NADPH oxydase (PHOX) activation especially in the case of HAM. In HAM, the maximal enhancing effects were obtained with monomethyl ramified saturated (MMRS) and linear unsaturated (LU) FAs such as

  20. OspF directly attenuates the activity of extracellular signal-regulated kinase during invasion by Shigella flexneri in human dendritic cells.

    Science.gov (United States)

    Kim, Dong Wook; Chu, Hyuk; Joo, Dong Hyun; Jang, Mi Seon; Choi, Jin Huk; Park, Sung-Moo; Choi, Yun-Jae; Han, Seung Hyun; Yun, Cheol-Heui

    2008-06-01

    Shigella spp., Gram-negative pathogenic bacteria, deliver various effector molecules into the host cell cytoplasm through their type III secretion system to facilitate their invasive process and control the host innate immune responses. Although the function of these effectors is well characterized in epithelial cells during Shigella infection, it has not been elucidated in the dendritic cell (DC), a major antigen presenting cell playing an important role in the initiation of immune responses. In this study, we showed that an invasive Shigella strain (M90T), but not its non-invasive counterpart strain (BS176) induced apoptotic cell death in the human monocyte-derived DCs. Confocal microscopy using a lysosome-associated membrane protein 2 specific antibody demonstrated that the M90T escaped from phagosomes 2h post-DC invasion while BS176 remained in the phagosome. Furthermore, Shigella expressed outer Shigella protein F (OspF), one of the effector proteins that are released through type III secretion system during the invasion, at non-secretion state and further up-regulated OspF expression in the cytoplasm of DC during the invasion. Interestingly, in the host cell, OspF could directly bind to the extracellular signal-regulated kinase (Erk) 1/2 and dephosphorylate phospho-Erk. These results suggest that induction of OspF is enhanced during Shigella invasion of DCs and decreases the phosphorylation level of Erk1/2, which could be at least partially involved in the apoptotic death of DC, eventually resulting in the down-regulation of the host immune response.

  1. The JAK kinase inhibitor CP-690,550 suppresses the growth of human polycythemia vera cells carrying the JAK2V617F mutation.

    Science.gov (United States)

    Manshouri, Taghi; Quintás-Cardama, Alfonso; Nussenzveig, Roberto H; Gaikwad, Amos; Estrov, Zeev; Prchal, Josef; Cortes, Jorge E; Kantarjian, Hagop M; Verstovsek, Srdan

    2008-06-01

    The somatic activating janus kinase 2 mutation (JAK2)(V617F) is detectable in most patients with polycythemia vera (PV). Here we report that CP-690,550 exerts greater antiproliferative and pro-apoptotic activity against cells harboring JAK2(V617F) compared with JAK2(WT). CP-690,550 treatment of murine factor-dependent cell Patersen-erythropoietin receptor (FDCP-EpoR) cells harboring human wild-type or V617F JAK2 resulted in inhibition of cell proliferation with a 50% inhibitory concentration (IC(50)) of 2.1 microM and 0.25 microM, respectively. Moreover, CP-690,550 induced a significant pro-apoptotic effect on murine FDCP-EpoR cells carrying JAK2(V617F), whereas a lesser effect was observed for cells carrying wild-type JAK2. This activity was coupled with inhibition of phosphorylation of the key JAK2(V617F)-dependent downstream signaling effectors signal transducer and activator of transcription (STAT)3, STAT5, and v-akt murine thymoma viral oncogene homolog (AKT). Furthermore, CP-690,550 treatment of ex-vivo-expanded erythroid progenitors from JAK2(V617F)-positive PV patients resulted in specific, antiproliferative (IC(50) = 0.2 microM) and pro-apoptotic activity. In contrast, expanded progenitors from healthy controls were less sensitive to CP-690,550 in proliferation (IC(50) > 1.0 microM), and apoptosis assays. The antiproliferative effect on expanded patient progenitors was paralleled by a decrease in JAK2(V617F) mutant allele frequency, particularly in a patient homozygous for JAK2(V617F). Flow cytometric analysis of expanded PV progenitor cells treated with CP-690,550 suggests a possible transition towards a pattern of erythroid differentiation resembling expanded cells from normal healthy controls.

  2. Measurement of Rho-Associated Kinase (ROCK) Activity in Humans: Validity of Leukocyte p-MBS/t-MBS in Comparison with Vascular Response to Fasudil

    Science.gov (United States)

    Hata, Takaki; Goto, Chikara; Soga, Junko; Hidaka, Takayuki; Fujii, Yuichi; Idei, Naomi; Fujimura, Noritaka; Maruhashi, Tatsuya; Mikami, Shinsuke; Kihara, Yasuki; Chayama, Kazuaki; Noma, Kensuke; Liao, James K.; Higashi, Yukihito

    2013-01-01

    Background Rho-associated kinases (ROCKs) have been shown to be involved in the pathogenesis of atherosclerosis. It is clinically important to estimate the degree of ROCK activity in humans. The purpose of this study was to confirm the validity of a leukocyte ROCK parameter as an index of ROCK activity in comparison with vascular response to a ROCK inhibitor. Methods and Results We evaluated the ratio of phospho myosin-binding subunit (p-MBS) on myosin light-chain phosphatase to total MBS in peripheral leukocytes by Western blot analysis and forearm blood flow (FBF) response to the ROCK inhibitor fasudil using strain-gauge plethysmography in 36 healthy subjects and 39 patients with cardiovascular diseases. Fasudil (3, 10, 30 μg/min) was infused intra-arterially for 5 minutes at each dose. Leukocyte p-MBS/total-MBS was higher in cardiovascular diseases than in healthy subjects (0.97±0.37 vs. 0.51±0.14; P=0.002). Fasudil increased FBF from 4.9±1.2 to 14.5±5.7 mL/min/100 mL tissue (P<0.0001) in patients with cardiovascular diseases, while fasudil did not alter FBF in healthy subjects. There was a significant relationship between leukocyte p-MBS/total-MBS and maximal FBF response to fasudil in all subjects (r=0.72, P<0.0001). There was also a significant correlation between p-MBS/total-MBS and maximal FBF response to fasudil in patients with cardiovascular diseases (r=0.59, P<0.0001). In healthy subjects, there was no significant correlation between the two parameters. Conclusions These findings suggest that assessment of leukocyte ROCK activity is minimally invasive and does not require pharmacologic intervention using ROCK inhibitors. Leukocyte p-MBS/total-MBS may be useful for evaluating ROCK activity in a clinical setting. PMID:21035804

  3. Prostaglandin E2 Inhibits Histamine-Evoked Ca2+ Release in Human Aortic Smooth Muscle Cells through Hyperactive cAMP Signaling Junctions and Protein Kinase A.

    Science.gov (United States)

    Taylor, Emily J A; Pantazaka, Evangelia; Shelley, Kathryn L; Taylor, Colin W

    2017-11-01

    In human aortic smooth muscle cells, prostaglandin E2 (PGE2) stimulates adenylyl cyclase (AC) and attenuates the increase in intracellular free Ca2+ concentration evoked by activation of histamine H1 receptors. The mechanisms are not resolved. We show that cAMP mediates inhibition of histamine-evoked Ca2+ signals by PGE2 Exchange proteins activated by cAMP were not required, but the effects were attenuated by inhibition of cAMP-dependent protein kinase (PKA). PGE2 had no effect on the Ca2+ signals evoked by protease-activated receptors, heterologously expressed muscarinic M3 receptors, or by direct activation of inositol 1,4,5-trisphosphate (IP3) receptors by photolysis of caged IP3 The rate of Ca2+ removal from the cytosol was unaffected by PGE2, but PGE2 attenuated histamine-evoked IP3 accumulation. Substantial inhibition of AC had no effect on the concentration-dependent inhibition of Ca2+ signals by PGE2 or butaprost (to activate EP2 receptors selectively), but it modestly attenuated responses to EP4 receptors, activation of which generated less cAMP than EP2 receptors. We conclude that inhibition of histamine-evoked Ca2+ signals by PGE2 occurs through "hyperactive signaling junctions," wherein cAMP is locally delivered to PKA at supersaturating concentrations to cause uncoupling of H1 receptors from phospholipase C. This sequence allows digital signaling from PGE2 receptors, through cAMP and PKA, to histamine-evoked Ca2+ signals. Copyright © 2017 by The Author(s).

  4. Resveratrol Modulates Interleukin-1β-induced Phosphatidylinositol 3-Kinase and Nuclear Factor κB Signaling Pathways in Human Tenocytes

    Science.gov (United States)

    Busch, Franziska; Mobasheri, Ali; Shayan, Parviz; Lueders, Cora; Stahlmann, Ralf; Shakibaei, Mehdi

    2012-01-01

    Resveratrol, an activator of histone deacetylase Sirt-1, has been proposed to have beneficial health effects due to its antioxidant and anti-inflammatory properties. However, the mechanisms underlying the anti-inflammatory effects of resveratrol and the intracellular signaling pathways involved are poorly understood. An in vitro model of human tenocytes was used to examine the mechanism of resveratrol action on IL-1β-mediated inflammatory signaling. Resveratrol suppressed IL-1β-induced activation of NF-κB and PI3K in a dose- and time-dependent manner. Treatment with resveratrol enhanced the production of matrix components collagen types I and III, tenomodulin, and tenogenic transcription factor scleraxis, whereas it inhibited gene products involved in inflammation and apoptosis. IL-1β-induced NF-κB and PI3K activation was inhibited by resveratrol or the inhibitors of PI3K (wortmannin), c-Src (PP1), and Akt (SH-5) through inhibition of IκB kinase, IκBα phosphorylation, and inhibition of nuclear translocation of NF-κB, suggesting that PI3K signaling pathway may be one of the signaling pathways inhibited by resveratrol to abrogate NF-κB activation. Inhibition of PI3K by wortmannin attenuated IL-1β-induced Akt and p65 acetylation, suggesting that p65 is a downstream component of PI3K/Akt in these responses. The modulatory effects of resveratrol on IL-1β-induced activation of NF-κB and PI3K were found to be mediated at least in part by the association between Sirt-1 and scleraxis and deacetylation of NF-κB and PI3K. Overall, these results demonstrate that activated Sirt-1 plays an essential role in the anti-inflammatory effects of resveratrol and this may be mediated at least in part through inhibition/deacetylation of PI3K and NF-κB. PMID:22936809

  5. Targeting of herpes simplex virus 1 thymidine kinase gene sequences into the OCT4 locus of human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Wu Ou

    Full Text Available The in vitro differentiation of human induced pluripotent stem cells (hiPSC to generate specific types of cells is inefficient, and the remaining undifferentiated cells may form teratomas. This raises safety concerns for clinical applications of hiPSC-derived cellular products. To improve the safety of hiPSC, we attempted to site-specifically insert a herpes simplex virus 1 thymidine kinase (HSV1-TK suicide gene at the endogenous OCT4 (POU5F1 locus of hiPSC. Since the endogenous OCT4 promoter is active in undifferentiated cells only, we speculated that the HSV1-TK suicide gene will be transcribed in undifferentiated cells only and that the remaining undifferentiated cells can be depleted by treating them with the prodrug ganciclovir (GCV prior to transplantation. To insert the HSV1-TK gene at the OCT4 locus, we cotransfected hiPSC with a pair of plasmids encoding an OCT4-specific zinc finger nuclease (ZFN and a donor plasmid harboring a promoter-less transgene cassette consisting of HSV1-TK and puromycin resistance gene sequences, flanked by OCT4 gene sequences. Puromycin resistant clones were established and characterized regarding their sensitivity to GCV and the site of integration of the HSV1-TK/puromycin resistance gene cassette. Of the nine puromycin-resistant iPSC clones analyzed, three contained the HSV1-TK transgene at the OCT4 locus, but they were not sensitive to GCV. The other six clones were GCV-sensitive, but the TK gene was located at off-target sites. These TK-expressing hiPSC clones remained GCV sensitive for up to 90 days, indicating that TK transgene expression was stable. Possible reasons for our failed attempt to selectively target the OCT4 locus are discussed.

  6. The SGK1 Kinase Inhibitor SI113 Sensitizes Theranostic Effects of the 64CuCl2 in Human Glioblastoma Multiforme Cells

    Directory of Open Access Journals (Sweden)

    Giada Catalogna

    2017-08-01

    Full Text Available Background/Aims: The importance of copper in the metabolism of cancer cells has been widely studied in the last 20 years and a clear-cut association between copper levels and cancer deregulation has been established. Copper-64, emitting positrons and β-radiations, is indicated for the labeling of a large number of molecules suitable for radionuclide imaging as well as radionuclide therapy. Glioblastoma multiforme (GBM is the CNS tumor with the worse prognosis, characterized by high number of recurrences and strong resistance to chemo-radio therapy, strongly affecting patients survival. We have recently discovered and studied the small molecule SI113, as inhibitor of SGK1, a serine/threonine protein kinase, that affects several neoplastic phenotypes and signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation, perturbs cell cycle progression and restores chemo-radio sensibility by modulating SGK1-related substrates. In the present paper we aim to characterize the combined effects of 64CuCl2 and SI113 on human GBM cell lines with variable p53 expression. Methods: Cell viability, cell death and stress/authopagic related pathways were then analyzed by FACS and WB-based assays, after exposure to SI113 and/or 64CuCl2. Results: We demonstrate here, that i 64CuCl2 is able to induce a time and dose dependent modulation of cell viability (with different IC50 values in highly malignant gliomas and that the co-treatment with SI113 leads to ii additive/synergistic effects in terms of cell death; iii enhancement of the effects of ionizing radiations, probably by a TRC1 modulation; iv modulation of the autophagic response. Conclusions: Evidence reported here underlines the therapeutic potential of the combined treatment with SI113 and 64CuCl2 in GBM cells.

  7. WNT6 promotes the migration and differentiation of human dental pulp cells partly through c-Jun N-terminal kinase signaling pathway.

    Science.gov (United States)

    Li, Ruimin; Wang, Chenglin; Tong, Juan; Su, Yingying; Lin, Yunfeng; Zhou, Xuedong; Ye, Ling

    2014-07-01

    During the dental pulp repair process, human dental pulp cells (HDPCs) migrate to injury sites where they may differentiate into odontoblastlike cells. WNT6 plays a role in dental development and can activate a noncanonical pathway including the c-Jun N-terminal kinase (JNK) pathway. The mechanism of WNT6 in dental pulp repair is still unknown. The purpose of this study was to explore the potential role of the WNT6/JNK signaling pathway in the promotion of cell migration and the differentiation of HDPCs. The third passage of HDPCs were cultured in vitro and treated with WNT6 conditioned medium with or without the pretreatment of JNK inhibitor SP600125. The activation of JNK was detected by Western blot, the expression of c-Jun was quantified by reverse-transcription polymerase chain reaction, the migration of HDPCs was determined by wound healing and transwell migration assays, and the differentiation of HDPCs was investigated using alkaline phosphatase staining and alizarin red staining. The expression of odontogenesis-related genes such as Runt-related transcription factor 2, dentin sialophosphoprotein, and dentin matrix protein 1 was quantified. WNT6 activates the JNK pathway in HDPCs and enhances cell migration, mineralization nodule formation, and alkaline phosphatase activation. WNT6 also increases the expression of Runt-related transcription factor 2, dentin sialophosphoprotein, and dentin matrix protein messenger RNA in HDPCs. Blockage of the JNK pathway in HDPCs decreases but does not completely abolish the cell migration and differentiation capacity induced by WNT6. WNT6 activates the JNK signaling pathway in HDPCs, leading to migration and differentiation. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  8. LEFTY2 Controls Migration of Human Endometrial Cancer Cells via Focal Adhesion Kinase Activity (FAK) and miRNA-200a.

    Science.gov (United States)

    Alowayed, Nour; Salker, Madhuri S; Zeng, Ni; Singh, Yogesh; Lang, Florian

    2016-01-01

    LEFTY2, a suppressor of cell proliferation, tumor growth, regulator of stemness and embryonic differentiation, is a negative regulator of cancer cell reprogramming. Malignant transformation may lead to migration requiring loss of adhesion and gain of migratory activity. Signaling involved in the orchestration of migration, proliferation and spreading of cells include focal adhesion kinase (FAK) and adhesion molecule E-cadherin. The present study explored whether LEFTY2 influences the proliferation marker MKi67, FAK activity, E-cadherin abundance and migration of Ishikawa human endometrial carcinoma cells. Moreover, the study explored the involvement of microRNA-200a (miR-200a), which is known to regulate cellular adhesion by targeting E-Cadherin. FAK activity was estimated from FAK phosphorylation quantified by Western blotting, migration utilizing a wound healing assay, miR-200a and MKi67 expression levels utilizing qRT-PCR, cell proliferation and apoptosis using BrdU and Annexin V staining, respectively, and E-Cadherin (E-Cad) abundance, using confocal microscopy. LEFTY2 (25 ng/ml, 48 hours) treatment was followed by decrease of MKi67 expression, FAK activity and migration. LEFTY2 upregulated miRNA-200a and E-Cad protein level in Ishikawa cells. The effect of LEFTY2 on migration was mimicked by FAK inhibitor PF 573228 (50 µM). Addition of LEFTY2 in the presence of PF-573228 did not result in a further significant decline of migration. In conclusion, LEFTY2 down-regulates MKi67 expression and FAK activity, up-regulates miR-200a and E-cadherin, and is thus a powerful negative regulator of endometrial cell proliferation and migration. © 2016 The Author(s) Published by S. Karger AG, Basel.

  9. Broncho Vaxom (OM-85) modulates rhinovirus docking proteins on human airway epithelial cells via Erk1/2 mitogen activated protein kinase and cAMP.

    Science.gov (United States)

    Roth, Michael; Pasquali, Christian; Stolz, Daiana; Tamm, Michael

    2017-01-01

    Bronchial epithelial cells (BEC) are primary target for Rhinovirus infection through attaching to cell membrane proteins. OM-85, a bacterial extract, improves recovery of asthma and COPD patients after viral infections, but only part of the mechanism was addressed, by focusing on defined immune cells. We therefore determined the effect of OM-85 on isolated primary human BEC of controls (n = 8), asthma patients (n = 10) and COPD patients (n = 9). BEC were treated with OM-85 alone (24 hours) or infected with Rhinovirus. BEC survival was monitored by manual cell counting and Rhinovirus replication by lytic activity. Immuno-blotting and ELISA were used to determine the expression of Rhinovirus interacting proteins: intracellular adhesion molecule (ICAM), major histocompatibility complex class II (MHC-2), complement component C1q receptor (C1q-R), inducible T-Cell co-stimulator (ICOS), its ligand ICOSL, and myeloid differentiation primary response gene 88 (Myd88); as well as for signal transducers Erk1/2, p38, JNK mitogen activated protein kinases MAPK), and cAMP. OM-85 significantly reduced Rhinovirus-induced BEC death and virus replication. OM-85 significantly increased the expression of virus interacting proteins C1q-R and β-defensin in all 3 probes and groups, which was prevented by either Erk1/2 MAPK or cAMP inhibition. In addition, OM-85 significantly reduced Rhinovirus induced expression of ICAM1 involving p38 MAPK. In BEC OM-85 had no significant effect on the expression of ICOS, ICOSL and MHC-2 membrane proteins nor on the adaptor protein MyD88. The OM-85-induced increased of C1q-R and β-defensin, both important for antigen presentation and phagocytosis, supports its activity in host cell's defence against Rhinovirus infection.

  10. Broncho Vaxom (OM-85 modulates rhinovirus docking proteins on human airway epithelial cells via Erk1/2 mitogen activated protein kinase and cAMP.

    Directory of Open Access Journals (Sweden)

    Michael Roth

    Full Text Available Bronchial epithelial cells (BEC are primary target for Rhinovirus infection through attaching to cell membrane proteins. OM-85, a bacterial extract, improves recovery of asthma and COPD patients after viral infections, but only part of the mechanism was addressed, by focusing on defined immune cells.We therefore determined the effect of OM-85 on isolated primary human BEC of controls (n = 8, asthma patients (n = 10 and COPD patients (n = 9.BEC were treated with OM-85 alone (24 hours or infected with Rhinovirus. BEC survival was monitored by manual cell counting and Rhinovirus replication by lytic activity. Immuno-blotting and ELISA were used to determine the expression of Rhinovirus interacting proteins: intracellular adhesion molecule (ICAM, major histocompatibility complex class II (MHC-2, complement component C1q receptor (C1q-R, inducible T-Cell co-stimulator (ICOS, its ligand ICOSL, and myeloid differentiation primary response gene 88 (Myd88; as well as for signal transducers Erk1/2, p38, JNK mitogen activated protein kinases MAPK, and cAMP.OM-85 significantly reduced Rhinovirus-induced BEC death and virus replication. OM-85 significantly increased the expression of virus interacting proteins C1q-R and β-defensin in all 3 probes and groups, which was prevented by either Erk1/2 MAPK or cAMP inhibition. In addition, OM-85 significantly reduced Rhinovirus induced expression of ICAM1 involving p38 MAPK. In BEC OM-85 had no significant effect on the expression of ICOS, ICOSL and MHC-2 membrane proteins nor on the adaptor protein MyD88.The OM-85-induced increased of C1q-R and β-defensin, both important for antigen presentation and phagocytosis, supports its activity in host cell's defence against Rhinovirus infection.

  11. Novel Library of Selenocompounds as Kinase Modulators

    Directory of Open Access Journals (Sweden)

    Carmen Sanmartín

    2011-07-01

    Full Text Available Although the causes of cancer lie in mutations or epigenic changes at the genetic level, their molecular manifestation is the dysfunction of biochemical pathways at the protein level. The 518 protein kinases encoded by the human genome play a central role in various diseases, a fact that has encouraged extensive investigations on their biological function and three dimensional structures. Selenium (Se is an important nutritional trace element involved in different physiological functions with antioxidative, antitumoral and chemopreventive properties. The mechanisms of action for selenocompounds as anticancer agents are not fully understood, but kinase modulation seems to be a possible pathway. Various organosulfur compounds have shown antitumoral and kinase inhibition effects but, in many cases, the replacement of sulfur by selenium improves the antitumoral effect of compounds. Although Se atom possesses a larger atomic volume and nucleophilic character than sulfur, Se can also formed interactions with aminoacids of the catalytic centers of proteins. So, we propose a novel chemical library that includes organoselenium compounds as kinase modulators. In this study thirteen selenocompounds have been evaluated at a concentration of 3 or 10 µM in a 24 kinase panel using a Caliper LabChip 3000 Drug Discover Platform. Several receptor (EGFR, IGFR1, FGFR1… and non-receptor (Abl kinases have been selected, as well as serine/threonine/lipid kinases (AurA, Akt, CDKs, MAPKs… implicated in main cancer pathways: cell cycle regulation, signal transduction, angiogenesis regulation among them. The obtained results showed that two compounds presented inhibition values higher than 50% in at least four kinases and seven derivatives selectively inhibited one or two kinases. Furthermore, three compounds selectively activated IGF-1R kinase with values ranging from −98% to −211%. In conclusion, we propose that the replacement of sulfur by selenium seems to be

  12. AMP-Activated Kinase (AMPK Activation by AICAR in Human White Adipocytes Derived from Pericardial White Adipose Tissue Stem Cells Induces a Partial Beige-Like Phenotype.

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    Omar Abdul-Rahman

    Full Text Available Beige adipocytes are special cells situated in the white adipose tissue. Beige adipocytes, lacking thermogenic cues, morphologically look quite similar to regular white adipocytes, but with a markedly different response to adrenalin. White adipocytes respond to adrenergic stimuli by enhancing lipolysis, while in beige adipocytes adrenalin induces mitochondrial biogenesis too. A key step in the differentiation and function of beige adipocytes is the deacetylation of peroxisome proliferator-activated receptor (PPARγ by SIRT1 and the consequent mitochondrial biogenesis. AMP-activated protein kinase (AMPK is an upstream activator of SIRT1, therefore we set out to investigate the role of AMPK in beige adipocyte differentiation using human adipose-derived mesenchymal stem cells (hADMSCs from pericardial adipose tissue. hADMSCs were differentiated to white and beige adipocytes and the differentiation medium of the white adipocytes was supplemented with 100 μM [(2R,3S,4R,5R-5-(4-Carbamoyl-5-aminoimidazol-1-yl-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate (AICAR, a known activator of AMPK. The activation of AMPK with AICAR led to the appearance of beige-like morphological properties in differentiated white adipocytes. Namely, smaller lipid droplets appeared in AICAR-treated white adipocytes in a similar fashion as in beige cells. Moreover, in AICAR-treated white adipocytes the mitochondrial network was more fused than in white adipocytes; a fused mitochondrial system was characteristic to beige adipocytes. Despite the morphological similarities bet