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Sample records for abelson tyrosine kinase

  1. Efficacy of the polo-like kinase inhibitor rigosertib, alone or in combination with Abelson tyrosine kinase inhibitors, against break point cluster region-c-Abelson-positive leukemia cells.

    Science.gov (United States)

    Okabe, Seiichi; Tauchi, Tetsuzo; Tanaka, Yuko; Sakuta, Juri; Ohyashiki, Kazuma

    2015-08-21

    The potency of Abelson (ABL) tyrosine kinase inhibitors (TKIs) against chronic myeloid leukemia (CML) has been demonstrated. However, ABL TKI resistance can develop. In this study, we investigated the efficacy of a combination therapy including rigosertib (ON 01910.Na), a polo-like kinase (PLK) and phosphoinositide 3-kinase (PI3K) inhibitor, and ABL TKIs. A 72-h rigosertib treatment was found to inhibit cell growth, induce apoptosis, reduce phosphorylation of the breakpoint cluster region-c (BCR)-ABL and its substrate Crk-L, and increase the activities of caspase 3 and poly (ADP-ribose) polymerase (PARP). This combination therapy also exerted a synergistic inhibitory effect on Philadelphia chromosome (Ph)-positive cell proliferation and reduced the phosphorylation of BCR-ABL and Crk-L while increasing that of cleaved PARP and the H2A.X histone. Rigosertib also potently inhibited the growth of ABL TKI-resistant cells, and cotreatment with ABL TKIs and rigosertib induced higher cytotoxicity. These results indicate that rigosertib treatment may be a powerful strategy against ABL TKI-resistant cells and could enhance the cytotoxic effects of ABL TKIs.

  2. In the absence of frazzled over-expression of Abelson tyrosine kinase disrupts commissure formation and causes axons to leave the embryonic CNS.

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    Joy N Dorsten

    Full Text Available BACKGROUND: In the Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra and the Abelson (Abl cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abl's role in attractive signaling, we over-expressed Abl in Fra mutants anticipating rescue of commissures. METHODOLOGY/PRINCIPAL FINDINGS: The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P-motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS. CONCLUSION/SIGNIFICANCE: Taken together, these data suggest that Fra activity is required to correctly regulate Abl-dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border.

  3. Tyrosine Kinase Inhibitors and Diabetes: A Novel Treatment Paradigm?

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    Fountas, Athanasios; Diamantopoulos, Leonidas-Nikolaos; Tsatsoulis, Agathocles

    2015-11-01

    Deregulation of protein tyrosine kinase (PTK) activity is implicated in various proliferative conditions. Multi-target tyrosine kinase inhibitors (TKIs) are increasingly used for the treatment of different malignancies. Recently, several clinical cases of the reversal of both type 1 and 2 diabetes mellitus (T1DM, T2DM) during TKI administration have been reported. Experimental in vivo and in vitro studies have elucidated some of the mechanisms behind this effect. For example, inhibition of Abelson tyrosine kinase (c-Abl) results in β cell survival and enhanced insulin secretion, while platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) inhibition leads to improvement in insulin sensitivity. In addition, inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) reduces the degree of islet cell inflammation (insulitis). Therefore, targeting several PTKs may provide a novel approach for correcting the pathophysiologic disturbances of diabetes.

  4. Tyrosine kinases in rheumatoid arthritis

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    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  5. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  6. Endocytosis of Receptor Tyrosine Kinases

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    Goh, Lai Kuan

    2013-01-01

    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

  7. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    phosphorylation. Protein-tyrosine phosphorylation in bacteria is particular with respect to very low occupancy of phosphorylation sites in vivo; this has represented a major challenge for detection techniques. Only the recent breakthroughs in gel-free high resolution mass spectrometry allowed the systematic...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  8. Receptor tyrosine kinases in carcinogenesis.

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    Zhang, Xiao-Ying; Zhang, Pei-Ying

    2016-11-01

    Receptor tyrosine kinases (RTKs) are cell surface glycoproteins with enzymatic activity involved in the regulation of various important functions. In all-important physiological functions including differentiation, cell-cell interactions, survival, proliferation, metabolism, migration and signaling these receptors are the key players of regulation. Additionally, mutations of RTKs or their overexpression have been described in many human cancers and are being explored as a novel avenue for a new therapeutic approach. Some of the deregulated RTKs observed to be significantly affected in cancers included vascular endothelial growth factor receptor, epidermal growth factor receptor, fibroblast growth factor receptor, RTK-like orphan receptor 1 (ROR1) and the platelet-derived growth factor receptor. These deregulated RTKs offer attractive possibilities for the new anticancer therapeutic approach involving specific targeting by monoclonal antibodies as well as kinase. The present review aimed to highlight recent perspectives of RTK ROR1 in cancer.

  9. TYROSINE KINASE INHIBITORS AND PREGNANCY

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    Elisabetta Abruzzese

    2014-04-01

    Full Text Available The management of patients with chronic myeloid leukemia (CML during pregnancy has became recently a matter of continuous debate.  The introduction of the Tyrosine Kinase Inhibitors (TKIs in clinical practice has dramatically changed the prognosis of CML patients.  Patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy.  This fact has come the necessity to address issues relating to fertility and pregnancy. Physicians are not infrequently being asked for advice regarding the need for, and or the appropriateness of, stopping treatment in order to conceive. In this report we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for all the approved TKIs, as well as suggest how to manage a planned and/or unplanned pregnancy.

  10. Cloning and expression of catalytic domain of Abl protein tyrosine kinase gene in E. coli

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiation and are involved in signal transduction. Uncontrolled signaling from receptor tyrosine kinases to intracellular tyrosine kinases can lead to inflamma tory responses and diseases such as cancer and atherosclerosis. Thus, inhibitors that block the activity of tyrosine kinases or the signaling pathways of PTKs activation could be assumed as the potential candidate for drug development. On this assumption, we cloned and expressed the Abl PTK gene in E. coli, and purified the PTK, which was used to screen the PTK inhibitors from the extracts of Chinese herbs. The catalytic domain sequence of PTK gene was amplified by PCR us ing the cDNA of abl from Abelson murine leukemia virus as template. The amplified fragment was then cloned into the GST-tagged expression vector pGEX2T. The recombinant plasmid was transformed into host cell E. coli DH5α and was induced to express PTK protein. The expression of the protein was detected using SDS-PAGE. The result showed that a specific protein was induced to express after 12 min induction, and reached peak level about 40% of the host total pro tein after 4 h induction. The molecular weight of the fusion protein was about 58 kD. The purified GST-PTK fusion pro tein presented higher activity for tyrosine phosphorylation.

  11. Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology.

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    Challa, Anil Kumar; Chatti, Kiranam

    2013-09-01

    Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome.

  12. Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases.

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    Chen, Sen; Jiang, Xinnong; Gewinner, Christina A; Asara, John M; Simon, Nicholas I; Cai, Changmeng; Cantley, Lewis C; Balk, Steven P

    2013-05-28

    The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr⁵⁷⁷ subsequent to its Src-mediated phosphorylation at Tyr⁵⁷⁶. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰, as well as Tyr¹¹⁸⁵, and downstream phosphorylation of the kinase AKT at Thr³⁰⁸ were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser⁴⁷³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases.

  13. Therapeutic Innovations: Tyrosine Kinase Inhibitors in Cancer

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    Nikolaos Dervisis

    2016-01-01

    Full Text Available Conventional cytotoxic chemotherapy involving DNA-interacting agents and indiscriminate cell death is no longer the future of cancer management. While chemotherapy is not likely to completely disappear from the armamentarium; the use of targeted therapies in combination with conventional treatment is becoming the standard of care in human medicine. Tyrosine kinases are pivotal points of functional cellular pathways and have been implicated in malignancy, inflammatory, and immune-mediated diseases. Pharmaceutical interventions targeting aberrant tyrosine kinase signaling has exploded and is the second most important area of drug development. The “Valley of Death” between drug discovery and approval threatens to blunt the enormous strides in cancer management seen thus far. Kinase inhibitors, as targeted small molecules, hold promise in the treatment and diagnosis of cancer. However, there are still many unanswered questions regarding the use of kinase inhibitors in the interpretation and management of cancer. Comparative oncology has the potential to address restrictions and limitations in the advancement in kinase inhibitor therapy.

  14. Tyrosine Kinase Display of Prostate Cancer Cells

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    2001-10-01

    Swanson, P.E., Ratliff, T.L., Vollmer, R.T., Day, M.L. (1995) HGF and its receptor c-met in prostatic carcinoma. Am. J. Pathol. 147:386-396. 4. Levitzki , A...alpha and erbB-3 receptor in human prostatic adenocarcinoma. Br. J. Urol. 79: 212-216. 78. Levitzki , A. and A. Gazit. 1995. Tyrosine kinase inhibition

  15. ABL Tyrosine Kinase Stimulates PUMA Protein Expression

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    Oon, Chet K

    2016-01-01

    ABL is an ubiquitously expressed non-receptor tyrosine kinase involved in multiple cellular functions including programmed cell death. Upon DNA damage, ABL has been shown to upregulate PUMA, p53 upregulated modulator of apoptosis, and causes downstream mitochondrial intrinsic apoptotic events. However, the mechanism by which ABL regulates PUMA expression remains unknown. We have shown that ABL does not change PUMA protein subcellular localization through immunofluorescence. Through protein an...

  16. Tyrosine Kinase Inhibition: An Approach to Drug Development

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    Levitzki, Alexander; Gazit, Aviv

    1995-03-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.

  17. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors: New Hope for Success in Multiple Sclerosis Therapy.

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    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak; Azizi, Gholamreza

    2014-07-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials.

  18. A secreted tyrosine kinase acts in the extracellular environment.

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    Bordoli, Mattia R; Yum, Jina; Breitkopf, Susanne B; Thon, Jonathan N; Italiano, Joseph E; Xiao, Junyu; Worby, Carolyn; Wong, Swee-Kee; Lin, Grace; Edenius, Maja; Keller, Tracy L; Asara, John M; Dixon, Jack E; Yeo, Chang-Yeol; Whitman, Malcolm

    2014-08-28

    Although tyrosine phosphorylation of extracellular proteins has been reported to occur extensively in vivo, no secreted protein tyrosine kinase has been identified. As a result, investigation of the potential role of extracellular tyrosine phosphorylation in physiological and pathological tissue regulation has not been possible. Here, we show that VLK, a putative protein kinase previously shown to be essential in embryonic development, is a secreted protein kinase, with preference for tyrosine, that phosphorylates a broad range of secreted and ER-resident substrate proteins. We find that VLK is rapidly and quantitatively secreted from platelets in response to stimuli and can tyrosine phosphorylate coreleased proteins utilizing endogenous as well as exogenous ATP sources. We propose that discovery of VLK activity provides an explanation for the extensive and conserved pattern of extracellular tyrosine phosphophorylation seen in vivo, and extends the importance of regulated tyrosine phosphorylation into the extracellular environment.

  19. Complexity of Receptor Tyrosine Kinase Signal Processing

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    Volinsky, Natalia; Kholodenko, Boris N.

    2013-01-01

    Our knowledge of molecular mechanisms of receptor tyrosine kinase (RTK) signaling advances with ever-increasing pace. Yet our understanding of how the spatiotemporal dynamics of RTK signaling control specific cellular outcomes has lagged behind. Systems-centered experimental and computational approaches can help reveal how overlapping networks of signal transducers downstream of RTKs orchestrate specific cell-fate decisions. We discuss how RTK network regulatory structures, which involve the immediate posttranslational and delayed transcriptional controls by multiple feed forward and feedback loops together with pathway cross talk, adapt cells to the combinatorial variety of external cues and conditions. This intricate network circuitry endows cells with emerging capabilities for RTK signal processing and decoding. We illustrate how mathematical modeling facilitates our understanding of RTK network behaviors by unraveling specific systems properties, including bistability, oscillations, excitable responses, and generation of intricate landscapes of signaling activities. PMID:23906711

  20. Asymmetric Tyrosine Kinase Arrangements in Activation or Autophosphorylation of Receptor Tyrosine Kinases

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    J Bae; J Schlessinger

    2011-12-31

    Receptor tyrosine kinases (RTKs) play important roles in the control of many cellular processes including cell proliferation, cell adhesion, angiogenesis, and apoptosis. Ligand-induced dimerization of RTKs leads to autophosphorylation and activation of RTKs. Structural studies have shown that while isolated ectodomains of several RTKs form symmetric dimers the isolated cytoplasmic kinase domains of epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) form asymmetric dimers during their activation. Binding of one kinase molecule of EGFR to a second kinase molecule asymmetrically leads to stimulation of kinase activity and enhanced autophosphorylation. Furthermore, the structures of the kinase domain of FGFR1 and FGFR2 reveal the formation of asymmetric interfaces in the processes of autophosphorylation at their specific phosphotyrosine (pY) sites. Disruption of asymmetric dimer interface of EGFR leads to reduction in enzymatic activity and drastic reduction of autophosphorylation of FGFRs in ligandstimulated live cells. These studies demonstrate that asymmetric dimer formation is as a common phenomenon critical for activation and autophosphorylation of RTKs.

  1. Skin problems and EGFR-tyrosine kinase inhibitor.

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    Kozuki, Toshiyuki

    2016-04-01

    Epidermal growth factor receptor inhibition is a good target for the treatment of lung, colon, pancreatic and head and neck cancers. Epidermal growth factor receptor-tyrosine kinase inhibitor was first approved for the treatment of advanced lung cancer in 2002. Epidermal growth factor receptor-tyrosine kinase inhibitor plays an essential role in the treatment of cancer, especially for patients harbouring epidermal growth factor receptor activating mutation. Hence, skin toxicity is the most concerning issue for the epidermal growth factor receptor-tyrosine kinase inhibitor treatment. Skin toxicity is bothersome and sometimes affects the quality of life and treatment compliance. Thus, it is important for physicians to understand the background and how to manage epidermal growth factor receptor-tyrosine kinase inhibitor-associated skin toxicity. Here, the author reviewed the mechanism and upfront preventive and reactive treatments for epidermal growth factor receptor inhibitor-associated skin toxicities.

  2. A novel proteomic approach for specific identification of tyrosine kinase substrates using [13C]tyrosine.

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    Ibarrola, Nieves; Molina, Henrik; Iwahori, Akiko; Pandey, Akhilesh

    2004-04-16

    Proteomic studies to find substrates of tyrosine kinases generally rely on identification of protein bands that are "pulled down" by antiphosphotyrosine antibodies from ligand-stimulated samples. One can obtain erroneous results from such experiments because of two major reasons. First, some proteins might be basally phosphorylated on tyrosine residues in the absence of ligand stimulation. Second, proteins can bind non-specifically to the antibodies or the affinity matrix. Induction of phosphorylation of proteins by ligand must therefore be confirmed by a different approach, which is not always feasible. We have developed a novel proteomic approach to identify substrates of tyrosine kinases in signaling pathways studies based on in vivo labeling of proteins with "light" (12C-labeled) or "heavy" (13C-labeled) tyrosine. This stable isotope labeling in cell culture method enables the unequivocal identification of tyrosine kinase substrates, as peptides derived from true substrates give rise to a unique signature in a mass spectrometry experiment. By using this approach, from a single experiment, we have successfully identified several known substrates of insulin signaling pathway and a novel substrate, polymerase I and transcript release factor, a protein that is implicated in the control of RNA metabolism and regulation of type I collagen promoters. This approach is amenable to high throughput global studies as it simplifies the specific identification of substrates of tyrosine kinases as well as serine/threonine kinases using mass spectrometry.

  3. Small molecule tyrosine kinase inhibitors in pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Sachin Gupta

    2008-10-01

    Full Text Available Sachin Gupta, Bassel F El-RayesDepartment of Hematology/Oncology, Karmanos Cancer Institute, Wayne State University, MI, USAAbstract: Pancreatic cancer has proven to be chemo-resistant, with gemcitabine being the only cytotoxic agent approved for advanced pancreatic cancer since 1996. Tyrosine kinase inhibitors represent a newer generation of chemotherapeutic agents targeting specific tumor pathways associated with carcinogenesis including cell cycle control, signal transduction, apoptosis and angiogenesis. These agents present a more selective way of treating pancreatic cancer. Erlotinib is the prototype of the tyrosine kinase inhibitors with proven efficacy in advanced pancreatic cancer and has been recently approved in that setting. Multiple other tyrosine kinase inhibitors targeting the VEGFR, PDGFR, and Src kinases are in various phases of clinical trials testing. The preliminary results of these trials have been disappointing. Current challenges in pancreatic cancer clinical trials testing include improving patient selection, identifying effective combinations, improving the predictive value of current preclinical models and better study designs. This review summarizes the present clinical development of tyrosine kinase inhibitors in pancreatic cancer and strategies for future drug development.Keywords: pancreatic cancer, erlotinib, tyrosine kinase inhibitors

  4. Tyrosine kinase blockers: new hope for successful cancer therapy.

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    Pytel, Dariusz; Sliwinski, Tomasz; Poplawski, Tomasz; Ferriola, Deborah; Majsterek, Ireneusz

    2009-01-01

    Tyrosine kinases (TKs) are attractive targets for cancer therapy, as quite often their abnormal signaling has been linked with tumor development and growth. Constitutive activated TKs stimulate multiple signaling pathways responsible for DNA repair, apoptosis, and cell proliferation. During the last few years, thorough analysis of the mechanism underlying tyrosine kinase's activity led to novel cancer therapy using TKs blockers. These drugs are remarkably effective in the treatment of various human tumors including head and neck, gastric, prostate and breast cancer and leukemias. The most successful example of kinase blockers is Imatinib (Imatinib mesylate, Gleevec, STI571), the inhibitor of Bcr/Abl oncoprotein, which has become a first-line therapy for chronic myelogenous leukemia. The introduction of STI571 for the treatment of leukemia in clinical oncology has had a dramatic impact on how this disease is currently managed. Others kinase inhibitors used recently in cancer therapy include Dasatinib (BMS-354825) specific for ABL non-receptor cytoplasmic kinase, Gefitinib (Iressa), Erlotinib (OSI-774, Tarceva) and Sunitinib (SU 11248, Sutent) specific for VEGF receptor kinase, AMN107 (Nilotinib) and INNO-406 (NS-187) specific for c-KIT kinase. The following TK blockers for treatment of various human tumors are in clinical development: Lapatinib (Lapatinib ditosylate, Tykerb, GW-572016), Canertinib (CI-1033), Zactima (ZD6474), Vatalanib (PTK787/ZK 222584), Sorafenib (Bay 43-9006, Nexavar), and Leflunomide (SU101, Arava). Herein, we discuss the chemistry, biological activity and clinical potential of new drugs with tyrosine kinase blockers for cancer treatment.

  5. Haemophilus ducreyi LspA proteins are tyrosine phosphorylated by macrophage-encoded protein tyrosine kinases.

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    Deng, Kaiping; Mock, Jason R; Greenberg, Steven; van Oers, Nicolai S C; Hansen, Eric J

    2008-10-01

    The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.

  6. Old Tyrosine Kinase Inhibitors and Newcomers in Gastrointestinal Cancer Treatment.

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    Giordani, Erika; Zoratto, Federica; Strudel, Martina; Papa, Anselmo; Rossi, Luigi; Minozzi, Marina; Caruso, Davide; Zaccarelli, Eleonora; Verrico, Monica; Tomao, Silverio

    2016-01-01

    Gastrointestinal cancer treatment is based more on molecular biology that has provided increasing knowledge about cancer pathogenesis on which targeted therapy is being developed. Precisely, targeted therapy is defined as a "type of treatment that uses drugs, such as monoclonal antibodies or tyrosine kinase inhibitors, to identify and attack specific cancer cells". Nowadays, the United States Food and Drug Administration has approved many targeted therapies for gastrointestinal cancer treatment, as many are in various phases of development as well. In a previous review we discussed the main monoclonal antibodies used and studied in gastrointestinal cancer. In addition to monoclonal antibodies, tyrosine kinase inhibitors represent another class of targeted therapy and following the approval of imatinib for gastrointestinal stromal tumours, other tyrosine kinase inhibitors have been approved for gastrointestinal cancers treatment such as sunitinib, regoragenib, sorafenib and erlotinib. Moving forward, the purpose of this review is to focus on the efficacy data of main tyrosine kinase inhibitors commonly used in the personalized treatment of each gastrointestinal tumour and to provide a comprehensive overview about experimental targeted therapies ongoing in this setting.

  7. Drug-drug interactions with tyrosine-kinase inhibitors

    NARCIS (Netherlands)

    Croes, Sander; de Vries, Frank

    2014-01-01

    In their Review, Roelof van Leeuwen and colleagues1 recommend various dose adjustments during concomitant use of tyrosine-kinase inhibitors and drugs that inhibit or induce cytochrome P450 3A4 (CYP3A4).1 Most information is taken from the US Food and Drug Administration (FDA)'s drug label or the Eur

  8. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.

    2007-01-01

    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  9. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title ...Receptor tyrosine kinases and the regulation of macrophage activation. Authors Co

  10. Proteolytic activation of ETK/Bmx tyrosine kinase by caspases.

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    Wu, Y M; Huang, C L; Kung, H J; Huang, C Y

    2001-05-25

    Etk/Bmx is a member of the Btk/Tec family of kinases, which are characterized by having a pleckstrin homology domain at the N terminus, in addition to the Src homology 3 (SH3), SH2, and the catalytic domains, shared with the Src family kinases. Etk, or Btk kinases in general, has been implicated in the regulation of apoptosis. To test whether Etk is the substrate for caspases during apoptosis, in vitro translated [(35)S]methionine-labeled Etk was incubated with different apoptotic extracts and recombinant caspases, respectively. Results showed that Etk was proteolyzed in all conditions tested with identical cleavage patterns. Caspase-mediated cleavage of Etk generated a C-terminal fragment, containing the complete SH2 and tyrosine kinase domains, but without intact pleckstrin homology and SH3 domains. This fragment has 4-fold higher kinase activity than that of the full-length Etk. Ectopic expression of the C-terminal fragment of Etk sensitized the PC3 prostate cancer cells to apoptosis in response to apoptosis-inducing stimuli. The finding, together with an earlier report that Etk is potentially antiapoptotic, suggests that Etk may serve as an apoptotic switch, depending on the forms of Etk existing inside the cells. To our knowledge, this is the first case where the activity of a tyrosine kinase is induced by caspase cleavage.

  11. Spatial regulation of receptor tyrosine kinases in development and cancer

    OpenAIRE

    Casaletto, Jessica B.; McClatchey, Andrea I.

    2012-01-01

    During development and tissue homeostasis, patterns of cellular organization, proliferation, and movement are highly choreographed. Receptor tyrosine kinases (RTKs) play a critical role in establishing these patterns. Individual cells and tissues exhibit tight spatial control of the RTKs that they express, enabling tissue morphogenesis and function while preventing unwarranted cell division and migration that can contribute to tumorigenesis. Indeed, RTKs are deregulated in most human cancers ...

  12. Eph-mediated tyrosine phosphorylation of citron kinase controls abscission

    Science.gov (United States)

    Jungas, Thomas; Perchey, Renaud T.; Fawal, Mohamad; Callot, Caroline; Froment, Carine; Burlet-Schiltz, Odile; Besson, Arnaud

    2016-01-01

    Cytokinesis is the last step of cell division, culminating in the physical separation of daughter cells at the end of mitosis. Cytokinesis is a tightly regulated process that until recently was mostly viewed as a cell-autonomous event. Here, we investigated the role of Ephrin/Eph signaling, a well-known local cell-to-cell communication pathway, in cell division. We show that activation of Eph signaling in vitro leads to multinucleation and polyploidy, and we demonstrate that this is caused by alteration of the ultimate step of cytokinesis, abscission. Control of abscission requires Eph kinase activity, and Src and citron kinase (CitK) are downstream effectors in the Eph-induced signal transduction cascade. CitK is phosphorylated on tyrosines in neural progenitors in vivo, and Src kinase directly phosphorylates CitK. We have identified the specific tyrosine residues of CitK that are phosphorylated and show that tyrosine phosphorylation of CitK impairs cytokinesis. Finally, we show that, similar to CitK, Ephrin/Eph signaling controls neuronal ploidy in the developing neocortex. Our study indicates that CitK integrates intracellular and extracellular signals provided by the local environment to coordinate completion of cytokinesis. PMID:27551053

  13. Robust versatile tyrosine kinase assay for HTS in drug discovery

    Science.gov (United States)

    Deshpande, Sudhir S.; Mineyev, I.; Owicki, John C.

    1999-04-01

    A fluorescence polarization assay was developed as an alternative to the radiolabeled SPA assays currently used to monitor the activity of tyrosine kinases in drug discovery. The assay can be used with enzymes having substrate specificity similar to that of the insulin receptor, the EGF receptor and the Src kinase receptor enzymes. The assay is easy to configure in 96, 384 and 1536-well microplates in assay volumes ranging from (mu) L with minimal efforts. The reconstituted reagents are stable for up to 24 hr at ambient temperatures, thereby minimizing the need for replenishing the stock solutions during the course of a high-throughput screen. Because of the stability and equilibrium kinetics, the assay allows the user the luxury of scheduling the reading of plates any time up to 24 hr after the completion of the assay without substantial deterioration in the assay signal. The antibody and the tracer solutions can also be premixed and added as a preformed complex in a single step. The performance of the assay with the insulin receptor kinase is described. In addition, given the diversity of the substrates used in measuring the activity of different tyrosine kinases, LJL's on-going efforts to provide different antibodies of wide ranging specificity and sensitivity are described.

  14. BRET biosensor analysis of receptor tyrosine kinase functionality

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    Sana eSiddiqui

    2013-04-01

    Full Text Available Bioluminescence resonance energy transfer (BRET is an improved version of earlier resonance energy transfer technologies used for the analysis of biomolecular protein interaction. BRET analysis can be applied to many transmembrane receptor classes, however the majority of the early published literature on BRET has focused on G protein-coupled receptor (GPCR research. In contrast, there is limited scientific literature using BRET to investigate receptor tyrosine kinase (RTK activity. This limited investigation is surprising as RTKs often employ dimerization as a key factor in their activation, as well as being important therapeutic targets in medicine, especially in the cases of cancer, diabetes, neurodegenerative and respiratory conditions. In this review, we consider an array of studies pertinent to RTKs and other non-GPCR receptor protein-protein signaling interactions; more specifically we discuss receptor-protein interactions involved in the transmission of signaling communication. We have provided an overview of functional BRET studies associated with the receptor tyrosine kinase (RTK super family involving: neurotrophic receptors (e.g. tropomyosin-related kinase (Trk and p75 neurotrophin receptor (p75NTR; insulinotropic receptors (e.g. insulin receptor (IR and insulin-like growth factor receptor (IGFR and growth factor receptors (e.g. ErbB receptors including the EGFR, the fibroblast growth factor receptor (FGFR, the vascular endothelial growth factor receptor (VEGFR and the c-kit and platelet-derived growth factor receptor (PDGFR. In addition, we review BRET-mediated studies of other tyrosine kinase-associated receptors including cytokine receptors, i.e. leptin receptor (OB-R and the growth hormone receptor (GHR. It is clear even from the relatively sparse experimental RTK BRET evidence that there is tremendous potential for this technological application for the functional investigation of RTK biology.

  15. Current understanding of tyrosine kinase BMX in inflammation and its inhibitors

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    Le Qiu

    2014-07-01

    Full Text Available Tec family kinases, which include tyrosine kinase expressed in hepatocellular carcinoma (TEC, Bruton's tyrosine kinase (BTK, interleukin (IL-2-inducible T-cell kinase (ITK, tyrosine-protein kinase (TXK, and bone marrow tyrosine kinase on chromosome X (BMX, are the second largest group of non-receptor tyrosine kinases and have a highly conserved carboxyl-terminal kinase domain. BMX was identified in human bone marrow cells, and was demonstrated to have been expressed in myeloid hematopoietic lineages cells, endothelial cells, and several types of cancers. Significant progress in this area during the last decade revealed an important role for BMX in inflammation and oncologic disorders. This review focuses on BMX biology, its role in inflammation and possible signaling pathways, and the potential of selective BMX inhibitors.

  16. Current understanding of tyrosine kinase BMX in inflammation and its inhibitors.

    Science.gov (United States)

    Qiu, Le; Wang, Fei; Liu, Sheng; Chen, Xu-Lin

    2014-01-01

    Tec family kinases, which include tyrosine kinase expressed in hepatocellular carcinoma (TEC), Bruton's tyrosine kinase (BTK), interleukin (IL)-2-inducible T-cell kinase (ITK), tyrosine-protein kinase (TXK), and bone marrow tyrosine kinase on chromosome X (BMX), are the second largest group of non-receptor tyrosine kinases and have a highly conserved carboxyl-terminal kinase domain. BMX was identified in human bone marrow cells, and was demonstrated to have been expressed in myeloid hematopoietic lineages cells, endothelial cells, and several types of cancers. Significant progress in this area during the last decade revealed an important role for BMX in inflammation and oncologic disorders. This review focuses on BMX biology, its role in inflammation and possible signaling pathways, and the potential of selective BMX inhibitors.

  17. Receptor tyrosine kinases and schistosome reproduction: new targets for chemotherapy

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    Marion eMorel

    2014-07-01

    Full Text Available Schistosome parasites still represent a serious public health concern and a major economic problem in developing countries. Pathology of schistosomiasis is mainly due to massive egg production by these parasites and to inflammatory responses raised against the eggs which are trapped in host tissues. Tyrosine kinases (TKs are key molecules that control cell differentiation and proliferation and they already represent important targets in cancer therapy. During the recent years, it has been shown that receptor tyrosine kinases (RTK signaling was active in reproductive organs and that it could regulate sexual maturation of schistosomes and egg production. This opens interesting perspectives for the control of transmission and pathogenesis of schistosomiasis based on new therapies targeting schistosome RTKs. This review relates the numerous data showing the major roles of kinase signaling in schistosome reproduction. It describes the conserved and particular features of schistosome RTKs, their implication in gametogenesis and reproduction processes and summarizes recent works indicating that RTKs and their signaling partners are interesting chemotherapeutical targets in new programs of control.

  18. Genomic organization of Bruton`s tyrosine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Rohrer, J.; Conley, M.E. [Univ. of Tennessee, Memphis, TN (United States)

    1994-09-01

    Bruton`s tyrosine kinase (Btk), is a nonreceptor tyrosine kinase that has been identified as the defective gene in X-linked agammaglobulinemia (XLA). XLA patients have profound hypogammaglobulinemia and markedly reduced numbers of B cells while their T cell and phagocyte numbers remain normal. To determine the genomic organization of Btk, intron/exon borders were identified by sequencing cosmid DNA using cDNA primers. Nineteen exons spanning 37 kb of genomic DNA were identified. All the intron/exon splice junctions followed the GT/AG rule. The translational ATG start codon was in exon 2 which was 6 kb downstream of exon 1. Exon 19, 519 bp in length and 3.8 kb distal to exon 18, was the largest exon and included the 450 bp of the 3{prime} untranslated region. Exons 6 through 18 formed the largest cluster of exons with no intron being longer than 1550 bp. There was no apparent correlation between the exon boundaries of Btk and the functional domains of the protein or the exon boundaries of src, the nonreceptor protein tyrosine kinase prototype. The region 500 bp upstream of the presumed transcriptional start site was sequenced and found to have a G+C content of 52%. No TATA-type promoter elements in the -20 bp to -30 bp region were identified. However, at position -48 bp, a TGTGAA motif was found that bears some similarity to the TATA box. This sequence was preceded by a perfect inverted CCAAT box at position -90 bp. Three retinoic acid binding sites were also identified at positions -50 bp, -83 bp and -197 bp. Defining the genomic structure of Btk will permit us to identify regulatory elements in this gene and to identify mutations in genomic DNA of patients with XLA.

  19. ABNORMAL PROTEIN TYROSINE KINASES ASSOCIATED WITH HUMAN HAEMATOLOGICAL MALIGNANCIES

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective: To survey the role of protein tyrosine kinases (PTKs) in the pathogenesis of several hematopoietic malignancies. Methods: By reviewing the published laboratory and clinical studies on PTK-related oncoproteins and their causative role in some leukemias and lymphomas. Results: Protein tyrosine kinases are key participants in signal transduction pathways that regulate cellular growth, activation and differentiations. Aberrant PTK activity resulting from gene mutation (often accompanying chromosome translocation) plays an etiologic role in several clonal hematopoietic malignancies. For example, the PTK product of the BCR-ABL fusion gene resulting from the t (9; 22) translocation exhibits several fold higher tyrosine kinase activity than the product of the ABL gene. Evidence suggests that the BCR-ABL oncoprotein alone is sufficient to case chronic myelogenous leukemia (CML) and other Ph positive acute leukemia. PTK over-activity resulting from chromosomal translocations creating TEL-ABL, TEL-JAK2 and TEL-PDGFR( fusion proteins plays an important role in the pathogenesis of other types of leukemia. Another example occurs in anaplastic large cell lymphoma (ALCL). Experimental and clinical evidences indicate that translocations involving ALK gene on chromosome 2p23, most commonly resulting in an NPM-ALK fusion oncogene, result in constitutive activation of ALK and cause ALCL. This group of lymphomas is now named ALK positive lymphoma or ALKoma. Conclusion: Genetic lesions creating aberrant fusion proteins that result in excessive PTK activity are increasingly being recognized as central to the pathogenesis of hemotopoietic malignancies. These chimeric PTK molecules represent attractive disease-specific targets against which new classes therapeutic agents are being developed.

  20. Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma

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    Wen-Bin Ou

    2011-01-01

    Full Text Available The receptor tyrosine kinases (RTKs epidermal growth factor receptor (EGFR and MET are activated in subsets of mesothelioma, suggesting that these kinases might represent novel therapeutic targets in this notoriously chemotherapy-resistant cancer. However, clinical trials have shown little activity for EGFR inhibitors in mesothelioma. Despite the evidence for RTK activation in mesothelioma pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. Using phospho-RTK and immunoblot assays, we herein demonstrate activation of multiple RTKs (EGFR, MET, AXL, and ERBB3 in individual mesothelioma cell lines but not in normal mesothelioma cells. Inhibition of mesothelioma multi-RTK signaling was accomplished using combinations of RTK direct inhibitors or by inhibition of the RTK chaperone, heat shock protein 90 (HSP90. Multi-RTK inhibition by the HSP90 inhibitor 17-allyloamino-17demethoxygeldanamycin (17-AAG had a substantially greater effect on mesothelioma proliferation and survival compared with inhibition of individual activated RTKs. HSP90 inhibition also suppressed phosphorylation of down-stream signaling intermediates (AKT, mitogen-activated protein kinase, and S6; upregulated the p53, p21, and p27 cell cycle checkpoints; induced G2 phase arrest; induced caspase 3/7 activity; and led to an increase in the sub-G1 apoptotic population. These compelling proapoptotic and antiproliferative responses indicate that HSP90 inhibition warrants clinical evaluation as a novel therapeutic strategy in mesothelioma.

  1. Analysis of receptor tyrosine kinase internalization using flow cytometry.

    Science.gov (United States)

    Li, Ning; Hill, Kristen S; Elferink, Lisa A

    2008-01-01

    The internalization of activated receptor tyrosine kinases (RTKs) by endocytosis and their subsequent down regulation in lysosomes plays a critical role in regulating the duration and intensity of downstream signaling events. Uncoupling of the RTK cMet from ligand-induced degradation was recently shown to correlate with sustained receptor signaling and increased cell tumorigenicity, suggesting that the corruption of these endocytic mechanisms could contribute to increased cMet signaling in metastatic cancers. To understand how cMet signaling for normal cell growth is controlled by endocytosis and how these mechanisms are dysregulated in metastatic cancers, we developed flow cytometry-based assays to examine cMet internalization.

  2. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

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    Belén Mezquita

    2014-02-01

    Full Text Available One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT and a truncated isoform of VEGFR-1 (i21-VEGFR-1, which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer.

  3. Function of Bruton's tyrosine kinase during B cell development is partially independent of its catalytic activity

    NARCIS (Netherlands)

    S. Middendorp; G.M. Dingjan (Gemma); A. Maas (Alex); K. Dahlenborg; R.W. Hendriks (Rudi)

    2003-01-01

    textabstractThe Tec family member Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes, an

  4. Tyrosine kinase inhibitors induced immune thrombocytopenia in chronic myeloid leukemia?

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    Avital F. Barak

    2011-12-01

    Full Text Available The outcome and quality of life of chronic myeloid leukemia (CML patients has remarkably changed with the treatment of tyrosine kinase inhibitors (TKIs. Currently, hematopoietic stem cell transplantation (HSCT is considered mainly as a third line salvage therapy in cases of TKIs resistance or intolerance. Here we describe a patient with chronic phase CML who developed both resistance and late occurrence of s severe thrombocytopenia on first and second generation TKIs and eventually underwent HSCT. Although the mechanism of the myelosuppression is not fully understood, we showed for the first time the development of dose dependent platelet antibodies in the presence of TKIs, suggesting the possibility of TKIs induced thrombocytopenia. Our case emphasizes that late development of severe myelosuppression during imatinib treatment is probably an important indication for consideration of early HSCT.

  5. Tyrosine Kinase Receptor Landscape in Lung Cancer: Therapeutical Implications

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    A. Quintanal-Villalonga

    2016-01-01

    Full Text Available Lung cancer is a heterogeneous disease responsible for the most cases of cancer-related deaths. The majority of patients are clinically diagnosed at advanced stages, with a poor survival rate. For this reason, the identification of oncodrivers and novel biomarkers is decisive for the future clinical management of this pathology. The rise of high throughput technologies popularly referred to as “omics” has accelerated the discovery of new biomarkers and drivers for this pathology. Within them, tyrosine kinase receptors (TKRs have proven to be of importance as diagnostic, prognostic, and predictive tools and, due to their molecular nature, as therapeutic targets. Along this review, the role of TKRs in the different lung cancer histologies, research on improvement of anti-TKR therapy, and the current approaches to manage anti-TKR resistance will be discussed.

  6. Precision Therapy for Lung Cancer: Tyrosine Kinase Inhibitors and Beyond.

    Science.gov (United States)

    Rajan, Arun; Schrump, David S

    2015-01-01

    For patients with advanced cancers there has been a concerted effort to transition from a generic treatment paradigm to one based on tumor-specific biologic, and patient-specific clinical characteristics. This approach, known as precision therapy has been made possible owing to widespread availability and a reduction in the cost of cutting-edge technologies that are used to study the genomic, proteomic, and metabolic attributes of individual tumors. This review traces the evolution of precision therapy for lung cancer from the identification of molecular subsets of the disease to the development and approval of tyrosine kinase, as well as immune checkpoint inhibitors for lung cancer therapy. Challenges of the precision therapy era including the emergence of acquired resistance, identification of untargetable mutations, and the effect on clinical trial design are discussed. We conclude by highlighting newer applications for the concept of precision therapy.

  7. SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyrosine kinase signaling.

    Science.gov (United States)

    Honorat, Jean-François; Ragab, Ashraf; Lamant, Laurence; Delsol, Georges; Ragab-Thomas, Jeannie

    2006-05-15

    Anaplastic large-cell lymphoma (ALCL) is frequently associated with the 2;5 translocation and expresses the NPM-ALK fusion protein, which possesses a constitutive tyrosine kinase activity. We analyzed SHP1 tyrosine phosphatase expression and activity in 3 ALK-positive ALCL cell lines (Karpas 299, Cost, and SU-DHL1) and in lymph node biopsies (n = 40). We found an inverse correlation between the level of NPM-ALK phosphorylation and SHP1 phosphatase activity. Pull-down and coimmunoprecipitation experiments demonstrated a SHP1/NPM-ALK association. Furthermore, confocal microscopy performed on ALCL cell lines and biopsy specimens showed the colocalization of the 2 proteins in cytoplasmic bodies containing Y664-phosphorylated NPM-ALK. Dephosphorylation of NPM-ALK by SHP1 demonstrated that NPM-ALK was a SHP1 substrate. Downregulation of SHP1 expression by RNAi in Karpas cells led to hyperphosphorylation of NPM-ALK, STAT3 activation, and increase in cell proliferation. Furthermore, SHP1 overexpression in 3T3 fibroblasts stably expressing NPM-ALK led to the decrease of NPM-ALK phosphorylation, lower cell proliferation, and tumor progression in nude mice. These findings show that SHP1 is a negative regulator of NPM-ALK signaling. The use of tissue microarrays revealed that 50% of ALK-positive ALCLs were positive for SHP1. Our results suggest that SHP1 could be a critical enzyme in ALCL biology and a potential therapeutic target.

  8. Mapping of p140Cap phosphorylation sites: the EPLYA and EGLYA motifs have a key role in tyrosine phosphorylation and Csk binding, and are substrates of the Abl kinase.

    Directory of Open Access Journals (Sweden)

    Daniele Repetto

    Full Text Available Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation. p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk, previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors.

  9. Mapping of p140Cap phosphorylation sites: the EPLYA and EGLYA motifs have a key role in tyrosine phosphorylation and Csk binding, and are substrates of the Abl kinase.

    Science.gov (United States)

    Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta; Sharma, Nanaocha; Grasso, Silvia; Russo, Isabella; Jensen, Ole N; Cabodi, Sara; Turco, Emilia; Di Stefano, Paola; Defilippi, Paola

    2013-01-01

    Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk), previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors.

  10. Influence of berberine on protein tyrosine kinase of erythrocyte insulin receptors from type 2 diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    Xianglei Deng; Xinrong Li; Chenggong Tian

    2005-01-01

    Objective: Bererine has been used to treat type 2 diabetes mellitus in Chinese traditional medicine because of its hypoglycemic effect. In this report, we compared the intrinsic tyrosine kinase activities of erythrocyte insulin receptors from type 2 diabetes mellitus with or without stimulation by berberine in vitro. Methods: Preparations containing insulin receptors were obtained from soluble human erythrocytes, and the insulin receptors were partially purified by affinity chromatography. The tyrosine kinase activity was measured by the exogenous substrate phosphorylation. Results: Both the membrane tyrosine kinase activity and the purified receptor tyrosine kinase activity from diabetics decreased significantly compared with those of normal individuals (reduced by 67.4 % and 47.2 %, respectively).After incubation with berberine, there is a statistical difference in the activity of membrane tyrosine kinase for diabetic patients (a 150% increase). Bererine had no effect on the tyrosine kinase activity of purified insulin receptors. Conclusion: We concluded from these results that berberine was able to improve the insulin sensitivity by increasing the protein tyrosine kinase activity of membrane-bound insulin receptors from type 2 diabetes mellitus.

  11. The Bmx tyrosine kinase induces activation of the Stat signaling pathway, which is specifically inhibited by protein kinase Cdelta.

    Science.gov (United States)

    Saharinen, P; Ekman, N; Sarvas, K; Parker, P; Alitalo, K; Silvennoinen, O

    1997-12-01

    Members of the hematopoietically expressed Tec tyrosine kinase family have an important role in hematopoietic signal transduction, as exemplified by the crucial role of Btk for B-cell differentiation and activation. Although a variety of cell surface receptors have been found to activate Tec tyrosine kinases, the specific signaling pathways and substrate molecules used by Tec kinases are still largely unknown. In this study a Tec family kinase, Bmx, was found to induce activation of the Stat signaling pathway. Bmx induced the tyrosine phosphorylation and DNA binding activity of all the Stat factors tested, including Stat1, Stat3, and Stat5, both in mammalian and insect cells. Bmx also induced transcriptional activation of Stat1- and Stat5-dependent reporter genes. Other cytoplasmic tyrosine kinases, Syk, Fyn, and c-Src, showed no or only weak ability to activate Stat proteins. Expression of Bmx in mammalian cells was found to induce activation of endogenous Stat proteins without activation of endogenous Jak kinases. We further analyzed the Bmx-mediated activation of Stat1, which was found to be regulated by protein kinase C delta (PKCdelta) isoform, but not beta 1, epsilon, or zeta isoforms, leading to inhibition of Stat1 tyrosine phosphorylation. In conclusion, these studies show that Bmx, a Tec family kinase, can function as an activator of the Stat signaling pathway and identify a role for PKCdelta in the regulation of Bmx signaling.

  12. Differential evolutionary wiring of the tyrosine kinase Btk.

    Directory of Open Access Journals (Sweden)

    Hossain M Nawaz

    Full Text Available BACKGROUND: A central question within biology is how intracellular signaling pathways are maintained throughout evolution. Btk29A is considered to be the fly-homolog of the mammalian Bruton's tyrosine kinase (Btk, which is a non-receptor tyrosine-kinase of the Tec-family. In mammalian cells, there is a single transcript splice-form and the corresponding Btk-protein plays an important role for B-lymphocyte development with alterations within the human BTK gene causing the immunodeficiency disease X-linked agammaglobulinemia in man and a related disorder in mice. In contrast, the Drosophila Btk29A locus encodes two splice-variants, where the type 2-form is the more related to the mammalian Btk gene product displaying more than 80% homology. In Drosophila, Btk29A displays a dynamic pattern of expression through the embryonic to adult stages. Complete loss-of-function of both splice-forms is lethal, whereas selective absence of the type 2-form reduces the adult lifespan of the fly and causes developmental abnormalities in male genitalia. METHODOLOGY/PRINCIPAL FINDINGS: Out of 7004-7979 transcripts expressed in the four sample groups, 5587 (70-79% were found in all four tissues and strains. Here, we investigated the role of Btk29A type 2 on a transcriptomic level in larval CNS and adult heads. We used samples either selectively defective in Btk29A type 2 (Btk29A(ficP or revertant flies with restored Btk29A type 2-function (Btk29A(fic Exc1-16. The whole transcriptomic profile for the different sample groups revealed Gene Ontology patterns reflecting lifespan abnormalities in adult head neuronal tissue, but not in larvae. CONCLUSIONS: In the Btk29A type 2-deficient strains there was no significant overlap between transcriptomic alterations in adult heads and larvae neuronal tissue, respectively. Moreover, there was no significant overlap of the transcriptomic changes between flies and mammals, suggesting that the evolutionary conservation is confined

  13. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  14. Role of Receptor Tyrosine Kinases and Their Ligands in Glioblastoma

    Directory of Open Access Journals (Sweden)

    Estefanía Carrasco-García

    2014-04-01

    Full Text Available Glioblastoma multiforme is the most frequent, aggressive and fatal type of brain tumor. Glioblastomas are characterized by their infiltrating nature, high proliferation rate and resistance to chemotherapy and radiation. Recently, oncologic therapy experienced a rapid evolution towards “targeted therapy,” which is the employment of drugs directed against particular targets that play essential roles in proliferation, survival and invasiveness of cancer cells. A number of molecules involved in signal transduction pathways are used as molecular targets for the treatment of various tumors. In fact, inhibitors of these molecules have already entered the clinic or are undergoing clinical trials. Cellular receptors are clear examples of such targets and in the case of glioblastoma multiforme, some of these receptors and their ligands have become relevant. In this review, the importance of glioblastoma multiforme in signaling pathways initiated by extracellular tyrosine kinase receptors such as EGFR, PDGFR and IGF-1R will be discussed. We will describe their ligands, family members, structure, activation mechanism, downstream molecules, as well as the interaction among these pathways. Lastly, we will provide an up-to-date review of the current targeted therapies in cancer, in particular glioblastoma that employ inhibitors of these pathways and their benefits.

  15. A novel putative tyrosine kinase receptor with oncogenic potential.

    Science.gov (United States)

    Janssen, J W; Schulz, A S; Steenvoorden, A C; Schmidberger, M; Strehl, S; Ambros, P F; Bartram, C R

    1991-11-01

    We have detected transforming activity by a tumorigenicity assay using NIH3T3 cells transfected with DNA from a chronic myeloproliferative disorder patient. Here, we report the cDNA cloning of the corresponding oncogene, designated UFO, in allusion to the as yet unidentified function of its protein. Nucleotide sequence analysis of a 3116bp cDNA clone revealed a 2682-bp-long open reading frame capable of directing the synthesis of a 894 amino acid polypeptide. The predicted UFO protein exhibits characteristic features of a transmembrane receptor with associated tyrosine kinase activity. The UFO proto-oncogene maps to human chromosome 19q13.1 and is transcribed into two 5.0 kb and 3.2 kb mRNAs in human bone marrow and human tumor cell lines. The UFO locus is evolutionarily conserved between vertebrate species. A 4.0 kb mRNA of the murine UFO homolog is expressed in a variety of different mouse tissues. We thus have identified a novel element of the complex signaling network involved in the control of cell proliferation and differentiation.

  16. Tyrosine inhibits creatine kinase activity in cerebral cortex of young rats.

    Science.gov (United States)

    de Andrade, Rodrigo Binkowski; Gemelli, Tanise; Rojas, Denise Bertin; Funchal, Cláudia; Dutra-Filho, Carlos Severo; Wannmacher, Clovis Milton Duval

    2011-09-01

    Tyrosine accumulates in inborn errors of tyrosine catabolism, especially in tyrosinemia type II, where tyrosine levels are highly elevated in tissues and physiological fluids of affected patients. Tyrosinemia type II is a disorder of autosomal recessive inheritance characterized by neurological symptoms similar to those observed in patients with creatine deficiency syndromes. Considering that the mechanisms of brain damage in these disorders are poorly known, in the present study our main objective was to investigate the in vivo and in vitro effects of different concentrations and preincubation times of tyrosine on cytosolic and mitochondrial creatine kinase activities of the cerebral cortex from 14-day-old Wistar rats. The cytosolic CK was reduced by 15% at 1 mM and 32% at 2 mM tyrosine. Similarly, the mitochondrial CK was inhibited by 15% at 1 mM and 22% at 2 mM tyrosine. We observed that the inhibition caused by tyrosine was concentration-dependent and was prevented by reduced glutathione. Results also indicated that mitochondrial, but not cytosolic creatine kinase activity was inhibited by tyrosine in a time-dependent way. Finally, a single injection of L-Tyrosine methyl ester administered i.p. decreased cytosolic (31%) and mitochondrial (18%) creatine kinase activities of brain cortex from rats. Considering that creatine kinase is an enzyme dependent of thiol residues for its function and tyrosine induces oxidative stress, the results suggest that the inhibition caused by tyrosine might occur by oxidation of essential sulfhydryl groups of the enzyme. In case this also occurs in patients with tyrosinemia, it is possible that creatine kinase inhibition may contribute to the neurological dysfunction characteristic of tyrosinemia.

  17. Functional Crosstalk Between WNT Signaling and Tyrosine Kinase Signaling in Cancer.

    Science.gov (United States)

    Anastas, Jaimie N

    2015-12-01

    Extensive molecular characterization of tumors has revealed that the activity of multiple signaling pathways is often simultaneously dampened or enhanced in cancer cells. Aberrant WNT signaling and tyrosine kinase signaling are two pathways that are frequently up- or downregulated in cancer. Although signaling pathways regulated by WNTs, tyrosine kinases, and other factors are often conceptualized as independent entities, the biological reality is likely much more complex. Understanding the mechanisms of crosstalk between multiple signal transduction networks is a key challenge for cancer researchers. The overall goals of this review are to describe mechanisms of crosstalk between WNT and tyrosine kinase pathways in cancer and to discuss how understanding intersections between WNT and tyrosine kinase signaling networks might be exploited to improve current therapies.

  18. Afatinib: emerging next-generation tyrosine kinase inhibitor for NSCLC

    Directory of Open Access Journals (Sweden)

    Nelson V

    2013-03-01

    Full Text Available Valerie Nelson, Jacqueline Ziehr, Mark Agulnik, Melissa JohnsonRobert H Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL, USAAbstract: The discovery of epidermal growth-factor receptor (EGFR-activating mutations and the introduction of oral EGFR tyrosine kinase inhibitors (EGFR-TKIs have expanded the treatment options for patients with non-small cell lung cancer. The first two reversible EGFR-TKIs, erlotinib and gefitinib, are approved for use in the first-line setting in patients with known EGFR-activating mutations and in the second- and third-line settings for all NSCLC patients. These first-generation EGFR-TKIs improve progression-free survival when compared to chemotherapy in patients with EGFR-activating mutations in the first-line setting. However, nearly all patients develop resistance to EGFR-directed agents. There is a need for further therapy options for patients with disease progression after treatment with reversible EGFR-TKIs. Afatinib is an irreversible ErbB family blocker that inhibits EGFR, HER2, and HER4. In vitro and in vivo, afatinib have shown increased inhibition of the common EGFR-activating mutations as well as the T790M resistance mutation when compared to erlotinib and gefitinib. Clinically, afatinib has been evaluated in the LUX-Lung series of trials, with improvement in progression-free survival reported in patients with EGFR-activating mutations in both first- and second-/third-line settings when compared to chemotherapy. Further investigation is needed to determine the precise role that afatinib will play in the treatment of patients with non-small cell lung cancer and EGFR-activating mutations.Keywords: afatinib, EGFR, irreversible EGFR inhibitor, EGPR-TKIs, LUX lung, resistance mutation, targeted therapy

  19. Mefloquine neurotoxicity is mediated by non-receptor tyrosine kinase.

    Science.gov (United States)

    Milatovic, Dejan; Jenkins, Jerry W; Hood, Jonathan E; Yu, Yingchun; Rongzhu, Lu; Aschner, Michael

    2011-10-01

    Among several available antimalarial drugs, mefloquine has proven to be effective against drug-resistant Plasmodium falciparum and remains the drug of choice for both therapy and chemoprophylaxis. However, mefloquine is known to cause adverse neurological and/or psychiatric symptoms, which offset its therapeutic advantage. The exact mechanisms leading to the adverse neurological effects of mefloquine are poorly defined. Alterations in neurotransmitter release and calcium homeostasis, the inhibition of cholinesterases and the interaction with adenosine A(2A) receptors have been hypothesized to play prominent roles in mediating the deleterious effects of this drug. Our recent data have established that mefloquine can also trigger oxidative damage and subsequent neurodegeneration in rat cortical primary neurons. Furthermore, we have utilized a system biology-centered approach and have constructed a pathway model of cellular responses to mefloquine, identifying non-receptor tyrosine kinase 2 (Pyk2) as a critical target in mediating mefloquine neurotoxicity. In this study, we sought to establish an experimental validation of Pyk2 using gene-silencing techniques (siRNA). We have examined whether the downregulation of Pyk2 in primary rat cortical neurons alters mefloquine neurotoxicity by evaluating cell viability, apoptosis and oxidative stress. Results from our study have confirmed that mefloquine neurotoxicity is associated with apoptotic response and oxidative injury, and we have demonstrated that mefloquine affects primary rat cortical neurons, at least in part, via Pyk2. The implication of these findings may prove beneficial in suppressing the neurological side effects of mefloquine and developing effective therapeutic modalities to offset its adverse effects.

  20. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Vanesa Olivares-Illana

    2008-06-01

    Full Text Available Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

  1. Identification of c-Src tyrosine kinase substrates using mass spectrometry and peptide microarrays

    DEFF Research Database (Denmark)

    Amanchy, Ramars; Zhong, Jun; Molina, Henrik;

    2008-01-01

    c-Src tyrosine kinase plays a critical role in signal transduction downstream of growth factor receptors, integrins and G protein-coupled receptors. We used stable isotope labeling with amino acids in cell culture (SILAC) approach to identify additional substrates of c-Src tyrosine kinase in human...... embryonic kidney 293T cells. We have identified 10 known substrates and interactors of c-Src and Src family kinases along with 26 novel substrates. We have experimentally validated 4 of the novel proteins (NICE-4, RNA binding motif 10, FUSE-binding protein 1 and TRK-fused gene) as direct substrates of c......-Src using in vitro kinase assays and cotransfection experiments. Significantly, using a c-Src specific inhibitor, we were also able to implicate 3 novel substrates (RNA binding motif 10, EWS1 and Bcl-2 associated transcription factor) in PDGF signaling. Finally, to identify the exact tyrosine residues...

  2. Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity.

    Science.gov (United States)

    Luz, Simão; Kongsuphol, Patthara; Mendes, Ana Isabel; Romeiras, Francisco; Sousa, Marisa; Schreiber, Rainer; Matos, Paulo; Jordan, Peter; Mehta, Anil; Amaral, Margarida D; Kunzelmann, Karl; Farinha, Carlos M

    2011-11-01

    Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

  3. Enhancement of cytosolic tyrosine kinase activity by propylthiouracil-induced hyperplasia in the rat thyroid.

    Science.gov (United States)

    Polychronakos, C; Piscina, R; Fantus, I G

    1989-01-01

    Hyperplasia of the thyroid gland induced by propylthiouracil (PTU) is a well established model of rapid cell proliferation in vivo. Recent evidence indicates that tyrosine kinase activity is associated with growth factor receptors and oncogene protein products and may have an important regulatory action in the control of cell growth. Thus, we examined tyrosine kinase activity in rat thyroid membrane and cytosol preparations at rest and during PTU-induced hyperplasia. Although kinase activity was present in a crude microsomal membrane preparation, no change was observed during thyroid growth. In contrast, tyrosine kinase activity assayed with the artificial substrate poly(Glu,Na:Tyr) 4:1 was present in normal rat thyroid cytosol and increased 2- to 6-fold during the rapid phase of hyperplasia in the first 5-10 days of PTU treatment. It declined to control values by day 15, when the size and DNA content of the thyroid reached a plateau. Preincubation of the cytosolic preparations with several peptides known to bind to and activate growth factor receptor tyrosine kinases failed to enhance the activity, suggesting, along with the cytosolic localization, that the activity was distinct from these receptors. By gel filtration chromatography and polyacrylamide gel electrophoresis, tyrosine kinase activity was associated with a 55 kDa protein. Partial purification over a poly(Glu,Na:Tyr)4:1-Sepharose column, yielded a protein that appeared capable of autophosphorylation. It is suggested that this tyrosine kinase plays a role in mediating the growth-promoting effects of this model of thyroid cell hyperplasia.

  4. Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate.

    Science.gov (United States)

    Wang, Qi; Vogan, Erik M; Nocka, Laura M; Rosen, Connor E; Zorn, Julie A; Harrison, Stephen C; Kuriyan, John

    2015-02-20

    Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

  5. Elucidation of a four-site allosteric network in fibroblast growth factor receptor tyrosine kinases

    Science.gov (United States)

    Chen, Huaibin; Marsiglia, William M; Cho, Min-Kyu; Huang, Zhifeng; Deng, Jingjing; Blais, Steven P; Gai, Weiming; Bhattacharya, Shibani; Neubert, Thomas A; Traaseth, Nathaniel J; Mohammadi, Moosa

    2017-01-01

    Receptor tyrosine kinase (RTK) signaling is tightly regulated by protein allostery within the intracellular tyrosine kinase domains. Yet the molecular determinants of allosteric connectivity in tyrosine kinase domain are incompletely understood. By means of structural (X-ray and NMR) and functional characterization of pathogenic gain-of-function mutations affecting the FGF receptor (FGFR) tyrosine kinase domain, we elucidated a long-distance allosteric network composed of four interconnected sites termed the ‘molecular brake’, ‘DFG latch’, ‘A-loop plug’, and ‘αC tether’. The first three sites repress the kinase from adopting an active conformation, whereas the αC tether promotes the active conformation. The skewed design of this four-site allosteric network imposes tight autoinhibition and accounts for the incomplete mimicry of the activated conformation by pathogenic mutations targeting a single site. Based on the structural similarity shared among RTKs, we propose that this allosteric model for FGFR kinases is applicable to other RTKs. DOI: http://dx.doi.org/10.7554/eLife.21137.001 PMID:28166054

  6. Lichen planopilaris-like eruption during treatment with tyrosine kinase inhibitor nilotinib*

    Science.gov (United States)

    Leitão, Juliana Ribeiro; Valente, Neusa Yuriko Sakai; Kakizaki, Priscila; Veronez, Isis Suga; Pires, Mario Cezar

    2016-01-01

    Tyrosine kinase inhibitors are effective as a target therapy for malignant neoplasms. Imatinib was the first tyrosine kinase inhibitor used. After its introduction, several other drugs have appeared with a similar mechanism of action, but less prone to causing resistance. Even though these drugs are selective, their toxicity does not exclusively target cancer cells, and skin toxicity is the most common non-hematologic adverse effect. We report an eruption similar to lichen planopilaris that developed during therapy with nilotinib, a second generation tyrosine kinase inhibitor, in a patient with chronic myeloid leukemia resistant to imatinib. In a literature review, we found only one report of non-scarring alopecia due to the use of nilotinib.

  7. The Cbl Proto-Oncogene Product Negatively Regulates the Src-Family Tyrosine Kinase Fyn by Enhancing Its Degradation

    OpenAIRE

    2000-01-01

    Fyn is a prototype Src-family tyrosine kinase that plays specific roles in neural development, keratinocyte differentiation, and lymphocyte activation, as well as roles redundant with other Src-family kinases. Similar to other Src-family kinases, efficient regulation of Fyn is achieved through intramolecular binding of its SH3 and SH2 domains to conserved regulatory regions. We have investigated the possibility that the tyrosine kinase regulatory protein Cbl provides a complementary mechanism...

  8. Using ovality to predict nonmutagenic, orally efficacious pyridazine amides as cell specific spleen tyrosine kinase inhibitors.

    Science.gov (United States)

    Lucas, Matthew C; Bhagirath, Niala; Chiao, Eric; Goldstein, David M; Hermann, Johannes C; Hsu, Pei-Yuan; Kirchner, Stephan; Kennedy-Smith, Joshua J; Kuglstatter, Andreas; Lukacs, Christine; Menke, John; Niu, Linghao; Padilla, Fernando; Peng, Ying; Polonchuk, Liudmila; Railkar, Aruna; Slade, Michelle; Soth, Michael; Xu, Daigen; Yadava, Preeti; Yee, Calvin; Zhou, Mingyan; Liao, Cheng

    2014-03-27

    Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.

  9. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar;

    2014-01-01

    . The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

  10. Tyrosine Phosphorylation of Tau by the Src Family Kinases Lck and Fyn

    Directory of Open Access Journals (Sweden)

    Bird Ian N

    2011-01-01

    Full Text Available Abstract Background Tau protein is the principal component of the neurofibrillary tangles found in Alzheimer's disease, where it is hyperphosphorylated on serine and threonine residues, and recently phosphotyrosine has been demonstrated. The Src-family kinase Fyn has been linked circumstantially to the pathology of Alzheimer's disease, and shown to phosphorylate Tyr18. Recently another Src-family kinase, Lck, has been identified as a genetic risk factor for this disease. Results In this study we show that Lck is a tau kinase. In vitro, comparison of Lck and Fyn showed that while both kinases phosphorylated Tyr18 preferentially, Lck phosphorylated other tyrosines somewhat better than Fyn. In co-transfected COS-7 cells, mutating any one of the five tyrosines in tau to phenylalanine reduced the apparent level of tau tyrosine phosphorylation to 25-40% of that given by wild-type tau. Consistent with this, tau mutants with only one remaining tyrosine gave poor phosphorylation; however, Tyr18 was phosphorylated better than the others. Conclusions Fyn and Lck have subtle differences in their properties as tau kinases, and the phosphorylation of tau is one mechanism by which the genetic risk associated with Lck might be expressed pathogenically.

  11. Phosphoinositide 3-kinase regulates crosstalk between Trk A tyrosine kinase and p75(NTR)-dependent sphingolipid signaling pathways.

    Science.gov (United States)

    Bilderback, T R; Gazula, V R; Dobrowsky, R T

    2001-03-01

    The mechanism of crosstalk between signaling pathways coupled to the Trk A and p75(NTR) neurotrophin receptors in PC12 cells was examined. In response to nerve growth factor (NGF), Trk A activation inhibited p75(NTR)-dependent sphingomyelin (SM) hydrolysis. The phosphoinositide 3-kinase (PI 3-kinase) inhibitor, LY294002, reversed this inhibition suggesting that Trk A activation of PI 3-kinase is necessary to inhibit sphingolipid signaling by p75(NTR). In contrast, SM hydrolysis induced by neurotrophin-3 (NT-3), which did not activate PI-3 kinase, was uneffected by LY294002. However, transient expression of a constituitively active PI 3-kinase inhibited p75(NTR)-dependent SM hydrolysis by both NGF and NT-3. Intriguingly, NGF induced an association of activated PI 3-kinase with acid sphingomyelinase (SMase). This interaction localized to caveolae-related domains and correlated with a 50% decrease in immunoprecipitated acid SMase activity. NGF-stimulated PI 3-kinase activity was necessary for inhibition of acid SMase but was not required for ligand-induced association of the p85 subunit of PI 3-kinase with the phospholipase. Finally, this interaction was specific for NGF since EGF did not induce an association of PI 3-kinase with acid SMase. In summary, our data suggest that PI 3-kinase regulates the inhibitory crosstalk between Trk A tyrosine kinase and p75(NTR)-dependent sphingolipid signaling pathways and that this interaction localizes to caveolae-related domains.

  12. Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells.

    Science.gov (United States)

    Sheremet, Ya A; Yemets, A I; Azmi, A; Vissenberg, K; Verbelen, J P; Blume, Ya B

    2012-01-01

    To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells.

  13. Sepsis-induced cardiac mitochondrial dysfunction involves altered mitochondrial-localization of tyrosine kinase Src and tyrosine phosphatase SHP2.

    Directory of Open Access Journals (Sweden)

    Qun S Zang

    Full Text Available Our previous research demonstrated that sepsis produces mitochondrial dysfunction with increased mitochondrial oxidative stress in the heart. The present study investigated the role of mitochondria-localized signaling molecules, tyrosine kinase Src and tyrosine phosphatase SHP2, in sepsis-induced cardiac mitochondrial dysfunction using a rat pneumonia-related sepsis model. SD rats were given an intratracheal injection of Streptococcus pneumoniae, 4×10(6 CFU per rat, (or vehicle for shams; heart tissues were then harvested and subcellular fractions were prepared. By Western blot, we detected a gradual and significant decrease in Src and an increase in SHP2 in cardiac mitochondria within 24 hours post-inoculation. Furthermore, at 24 hours post-inoculation, sepsis caused a near 70% reduction in tyrosine phosphorylation of all cardiac mitochondrial proteins. Decreased tyrosine phosphorylation of certain mitochondrial structural proteins (porin, cyclophilin D and cytochrome C and functional proteins (complex II subunit 30kD and complex I subunit NDUFB8 were evident in the hearts of septic rats. In vitro, pre-treatment of mitochondrial fractions with recombinant active Src kinase elevated OXPHOS complex I and II-III activity, whereas the effect of SHP2 phosphatase was opposite. Neither Src nor SHP2 affected complex IV and V activity under the same conditions. By immunoprecipitation, we showed that Src and SHP2 consistently interacted with complex I and III in the heart, suggesting that complex I and III contain putative substrates of Src and SHP2. In addition, in vitro treatment of mitochondrial fractions with active Src suppressed sepsis-associated mtROS production and protected aconitase activity, an indirect marker of mitochondrial oxidative stress. On the contrary, active SHP2 phosphatase overproduced mtROS and deactivated aconitase under the same in vitro conditions. In conclusion, our data suggest that changes in mitochondria

  14. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

    Directory of Open Access Journals (Sweden)

    Mili Jeon

    2012-04-01

    The respiratory (tracheal system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs, Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr tyrosine kinase (TK. Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways.

  15. DMPD: Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12472665 Macrophage-stimulating protein and RON receptor tyrosine kinase: potential...:545-53. (.png) (.svg) (.html) (.csml) Show Macrophage-stimulating protein and RON receptor tyrosine kinase:... potentialregulators of macrophage inflammatory activities. PubmedID 12472665 Title Macrophage-stim

  16. Reconstitution of Btk signaling by the atypical tec family tyrosine kinases Bmx and Txk.

    Science.gov (United States)

    Tomlinson, M G; Kurosaki, T; Berson, A E; Fujii, G H; Johnston, J A; Bolen, J B

    1999-05-07

    Bruton's tyrosine kinase (Btk) is mutated in X-linked agammaglobulinemia patients and plays an essential role in B cell receptor signal transduction. Btk is a member of the Tec family of nonreceptor protein-tyrosine kinases that includes Bmx, Itk, Tec, and Txk. Cell lines deficient for Btk are impaired in phospholipase C-gamma2 (PLCgamma2)-dependent signaling. Itk and Tec have recently been shown to reconstitute PLCgamma2-dependent signaling in Btk-deficient human cells, but it is not known whether the atypical Tec family members, Bmx and Txk, can reconstitute function. Here we reconstitute Btk-deficient DT40 B cells with Bmx and Txk to compare their function with other Tec kinases. We show that in common with Itk and Tec, Bmx reconstituted PLCgamma2-dependent responses including calcium mobilization, extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activation, and apoptosis. Txk also restored PLCgamma2/calcium signaling but, unlike other Tec kinases, functioned in a phosphatidylinositol 3-kinase-independent manner and failed to reconstitute apoptosis. These results are consistent with a common role for Tec kinases as amplifiers of PLCgamma2-dependent signal transduction, but suggest that the pleckstrin homology domain of Tec kinases, absent in Txk, is essential for apoptosis.

  17. Quinoxaline-Based Scaffolds Targeting Tyrosine Kinases and Their Potential Anticancer Activity.

    Science.gov (United States)

    El Newahie, Aliya M S; Ismail, Nasser S M; Abou El Ella, Dalal A; Abouzid, Khaled A M

    2016-05-01

    Quinoxaline derivatives, also called benzopyrazines, are an important class of heterocyclic compounds. Quinoxalines have drawn great attention due to their wide spectrum of biological activities. They are considered as an important basis for anticancer drugs due to their potential activity as protein kinase inhibitors. In this review, we focus on the chemistry of the quinoxaline derivatives, the strategies for their synthesis, their potential activities against various tyrosine kinases, and on the structure-activity relationship studies reported to date.

  18. Role of the Yes and Csk tyrosine kinases in the development of a pathological state in the human retina.

    Science.gov (United States)

    Baranova, Lyudmila; Emelyanova, Valentina; Volotovski, Igor

    2010-07-01

    Amplification and a cloning of fragments of genes of human retina tyrosine kinases, the nucleotide sequences of which feature a high homology to the gene families of the Yes and Csk tyrosine kinases, and a cloning of the complete coding sequence of the cDNA of the Csk tyrosine kinase gene of the human lymphocytes have been carried out. It has been established that this sequence contains 1,624 bp and encodes a protein that, with a 99% homology, corresponds to the human tyrosine kinase. A comparative analysis of the nucleotide sequences of the full-size cDNA of the Csk tyrosine kinase of the lymphocytes of healthy donors and of patients with an eye choroidal melanoma has shown that a risk of development of an eye choroidal melanoma can be estimated by the frequency of occurrence of a mutant allele in the 10th exon.

  19. Co-conserved features associated with cis regulation of ErbB tyrosine kinases.

    Directory of Open Access Journals (Sweden)

    Amar Mirza

    Full Text Available BACKGROUND: The epidermal growth factor receptor kinases, or ErbB kinases, belong to a large sub-group of receptor tyrosine kinases (RTKs, which share a conserved catalytic core. The catalytic core of ErbB kinases have functionally diverged from other RTKs in that they are activated by a unique allosteric mechanism that involves specific interactions between the kinase core and the flanking Juxtamembrane (JM and COOH-terminal tail (C-terminal tail. Although extensive studies on ErbB and related tyrosine kinases have provided important insights into the structural basis for ErbB kinase functional divergence, the sequence features that contribute to the unique regulation of ErbB kinases have not been systematically explored. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we use a Bayesian approach to identify the selective sequence constraints that most distinguish ErbB kinases from other receptor tyrosine kinases. We find that strong ErbB kinase-specific constraints are imposed on residues that tether the JM and C-terminal tail to key functional regions of the kinase core. A conserved RIxKExE motif in the JM-kinase linker region and a glutamine in the inter-lobe linker are identified as two of the most distinguishing features of the ErbB family. While the RIxKExE motif tethers the C-terminal tail to the N-lobe of the kinase domain, the glutamine tethers the C-terminal tail to hinge regions critical for inter-lobe movement. Comparison of the active and inactive crystal structures of ErbB kinases indicates that the identified residues are conformationally malleable and can potentially contribute to the cis regulation of the kinase core by the JM and C-terminal tail. ErbB3, and EGFR orthologs in sponges and parasitic worms, diverge from some of the canonical ErbB features, providing insights into sub-family and lineage-specific functional specialization. CONCLUSION/SIGNIFICANCE: Our analysis pinpoints key residues for mutational analysis, and

  20. Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling.

    Science.gov (United States)

    Holopainen, Tanja; Räsänen, Markus; Anisimov, Andrey; Tuomainen, Tomi; Zheng, Wei; Tvorogov, Denis; Hulmi, Juha J; Andersson, Leif C; Cenni, Bruno; Tavi, Pasi; Mervaala, Eero; Kivelä, Riikka; Alitalo, Kari

    2015-10-20

    Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy.

  1. Protein-Tyrosine Kinase Signaling in the Biological Functions Associated with Sperm

    Directory of Open Access Journals (Sweden)

    Takashi W. Ijiri

    2012-01-01

    Full Text Available In sexual reproduction, two gamete cells (i.e., egg and sperm fuse (fertilization to create a newborn with a genetic identity distinct from those of the parents. In the course of these developmental processes, a variety of signal transduction events occur simultaneously in each of the two gametes, as well as in the fertilized egg/zygote/early embryo. In particular, a growing body of knowledge suggests that the tyrosine kinase Src and/or other protein-tyrosine kinases are important elements that facilitate successful implementation of the aforementioned processes in many animal species. In this paper, we summarize recent findings on the roles of protein-tyrosine phosphorylation in many sperm-related processes (from spermatogenesis to epididymal maturation, capacitation, acrosomal exocytosis, and fertilization.

  2. Metabolite profiling of the multiple tyrosine kinase inhibitor lenvatinib : a cross-species comparison

    NARCIS (Netherlands)

    Dubbelman, Anne Charlotte; Nijenhuis, Cynthia M.; Jansen, Robert S; Rosing, Hilde; Mizuo, Hitoshi; Kawaguchi, Shinki; Critchley, David; Shumaker, Robert; Schellens, Jan H M; Beijnen, Jos H.

    2016-01-01

    Lenvatinib is an oral, multiple receptor tyrosine kinase inhibitor. Preclinical drug metabolism studies showed unique metabolic pathways for lenvatinib in monkeys and rats. A human mass balance study demonstrated that lenvatinib related material is mainly excreted via feces with a small fraction as

  3. Impaired precursor B cell differentiation in Bruton's tyrosine kinase-deficient mice

    NARCIS (Netherlands)

    S. Middendorp; G.M. Dingjan (Gemma); R.W. Hendriks (Rudi)

    2002-01-01

    textabstractBruton's tyrosine kinase (Btk) is a cytoplasmic signaling molecule that is crucial for precursor (pre-B) cell differentiation in humans. In this study, we show that during the transition of large cycling to small resting pre-B cells in the mouse, Btk-deficient cells fai

  4. Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L

    1994-01-01

    Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells...

  5. Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase

    DEFF Research Database (Denmark)

    Wang, X; Uhler, M D; Billestrup, N;

    1992-01-01

    The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels ...

  6. Tyrosine kinase inhibitors target cancer stem cells in renal cell cancer.

    Science.gov (United States)

    Czarnecka, Anna M; Solarek, Wojciech; Kornakiewicz, Anna; Szczylik, Cezary

    2016-03-01

    This study was designed to analyze the impact of multi-targeted tyrosine kinase inhibitors on the cancer stem cell subpopulation in renal cell cancer. The second objective was to evaluate the effect of tumor growth inhibition related to a tumor niche factor - oxygen deprivation - as hypoxia develops along with the anti-angiogenic activity of tyrosine kinase inhibitors in renal tumors. Cells were treated with tyrosine kinase inhibitors, sunitinib, sorafenib and axitinib, in 2D and 3D culture conditions. Cell proliferation along with drug toxicity were evaluated. It was shown that the proliferation rate of cancer stem cells was decreased by the tyrosine kinase inhibitors. The efficacy of the growth inhibition was limited by hypoxic conditions and 3D intratumoral cell-cell interactions. We conclude that understanding the complex molecular interaction feedback loops between differentiated cancer cells, cancer stem cells and the tumor microenvironment in 3D culture should aid the identification of novel treatment targets and to evalute the efficacy of renal cancer therapies. Cell-cell interaction may represent a critical microenvironmental factor regulating cancer stem cell self-renewal potential, enhancing the stem cell phenotype and limiting drug toxicity. At the same time the role of hypoxia in renal cancer stem cell biology is also significant.

  7. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus

    DEFF Research Database (Denmark)

    Olivares-Illana, Vanesa; Meyer, Philippe; Bechet, Emmanuelle

    2008-01-01

    understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus...

  8. Tyrosine Kinase Ligand-Receptor Pair Prediction by Using Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Masayuki Yarimizu

    2015-01-01

    Full Text Available Receptor tyrosine kinases are essential proteins involved in cellular differentiation and proliferation in vivo and are heavily involved in allergic diseases, diabetes, and onset/proliferation of cancerous cells. Identifying the interacting partner of this protein, a growth factor ligand, will provide a deeper understanding of cellular proliferation/differentiation and other cell processes. In this study, we developed a method for predicting tyrosine kinase ligand-receptor pairs from their amino acid sequences. We collected tyrosine kinase ligand-receptor pairs from the Database of Interacting Proteins (DIP and UniProtKB, filtered them by removing sequence redundancy, and used them as a dataset for machine learning and assessment of predictive performance. Our prediction method is based on support vector machines (SVMs, and we evaluated several input features suitable for tyrosine kinase for machine learning and compared and analyzed the results. Using sequence pattern information and domain information extracted from sequences as input features, we obtained 0.996 of the area under the receiver operating characteristic curve. This accuracy is higher than that obtained from general protein-protein interaction pair predictions.

  9. EphB4 Receptor Tyrosine Kinase in Prostate Cancer

    Science.gov (United States)

    2011-09-01

    Hassanieh  L,   Ley  EJ,  Scehnet  J,  Kumar  NG,   Hawes  D,  Press  MF,  Weaver  FA,  Gill  PS.  Receptor  tyrosine...J. Pathol. 174 (2009) 1492. [33] T.D. Bartley, R.W. Hunt, A.A. Welcher, W.J. Boyle , V.P. Parker, R.A. Lindberg, H.S. Lu, A.M. Colombero, R.L

  10. ACK1/TNK2 tyrosine kinase: molecular signaling and evolving role in cancers.

    Science.gov (United States)

    Mahajan, K; Mahajan, N P

    2015-08-01

    Deregulated tyrosine kinase signaling alters cellular homeostasis to drive cancer progression. The emergence of a non-receptor tyrosine kinase (non-RTK), ACK1 (also known as activated Cdc42-associated kinase 1 or TNK2) as an oncogenic kinase, has uncovered novel mechanisms by which tyrosine kinase signaling promotes cancer progression. Although early studies focused on ACK1 as a cytosolic effector of activated transmembrane RTKs, wherein it shuttles between the cytosol and the nucleus to rapidly transduce extracellular signals from the RTKs to the intracellular effectors, recent data unfold a new aspect of its functionality as an epigenetic regulator. ACK1 interacts with the estrogen receptor (ER)/histone demethylase KDM3A (JHDM2a) complex, which modifies KDM3A by tyrosine phosphorylation to regulate the transcriptional outcome at HOXA1 locus to promote the growth of tamoxifen-resistant breast cancer. It is also well established that ACK1 regulates the activity of androgen receptor (AR) by tyrosine phosphorylation to fuel the growth of hormone-refractory prostate cancers. Further, recent explosion in genomic sequencing has revealed recurrent ACK1 gene amplification and somatic mutations in a variety of human malignancies, providing a molecular basis for its role in neoplastic transformation. In this review, we will discuss the various facets of ACK1 signaling, including its newly uncovered epigenetic regulator function, which enables cells to bypass the blockade to major survival pathways to promote resistance to standard cancer treatments. Not surprisingly, cancer cells appear to acquire an 'addiction' to ACK1-mediated survival, particularly under stress conditions, such as growth factor deprivation or genotoxic insults or hormone deprivation. With the accelerated development of potent and selective ACK1 inhibitors, targeted treatment for cancers harboring aberrant ACK1 activity may soon become a clinical reality.

  11. Attenuation of endothelin-1-induced calcium response by tyrosine kinase inhibitors in vascular smooth muscle cells.

    Science.gov (United States)

    Liu, C Y; Sturek, M

    1996-06-01

    Although tyrosine kinases play an important role in cell growth and have been implicated in regulation of smooth muscle contraction, their role in agonist-induced myoplasmic Ca2+ responses is unclear. We examined effects of the tyrosine kinase inhibitors genistein and methyl 2,5-dihydroxycinnamate (MDHC) on the endothelin-1 (ET-1)-induced Ca2+ response and determined underlying mechanisms for the effects. Freshly isolated smooth muscle cells from porcine coronary arteries were loaded with fura 2 ester, and myoplasmic free Ca2+ (Ca2+ (m)) concentration was estimated with fura 2 microfluorometry. Both genistein and MDHC inhibited the initial transient Cam2+ response to ET by 54 and 81%, respectively (P latent period from ET-1 application to the beginning of the Cam2+ response being increased from 1.08 +/- 0.17 to 2.65 +/- 0.52 min (P < 0.05). In the absence of extracellular Ca2+, genistein inhibited the ET-1-induced Cam2+ response by 93% (P < 0.05). The Cam2+ responses to caffeine (5 mM) or inositol trisphosphate (IP3) applied intracellularly via a patch-clamp pipette were not affected by genistein. Both genistein and MDHC also abolished the sustained Cam2+ response to ET-1. However, the Cam2+ response to depolarization by 80 mM K+ was not inhibited by MDHC and only inhibited 22% by genistein (P < 0.05). These results indicate that 1) activation of tyrosine kinases is an important regulatory mechanism for the ET-1-induced Cam2+ response in vascular smooth muscle and 2) tyrosine kinases mediate ET-1-induced Ca2+ release with no direct effect on IP3-mediated Ca2+ release. Thus ET-1-mediated signaling upstream of IP3 interaction with the Ca2+ stores is regulated by tyrosine kinases.

  12. Masitinib (AB1010, a potent and selective tyrosine kinase inhibitor targeting KIT.

    Directory of Open Access Journals (Sweden)

    Patrice Dubreuil

    Full Text Available BACKGROUND: The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010, a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. METHODOLOGY/PRINCIPAL FINDINGS: In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC(50 of 200+/-40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC(50 of 150+/-80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. CONCLUSIONS: Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity.

  13. PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs).

    Science.gov (United States)

    Barrows, Douglas; He, John Z; Parsons, Ramon

    2016-09-16

    Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.

  14. Regulation of the EphA2 kinase by the low molecular weight tyrosine phosphatase induces transformation.

    Science.gov (United States)

    Kikawa, Keith D; Vidale, Derika R; Van Etten, Robert L; Kinch, Michael S

    2002-10-18

    Intracellular signaling by protein tyrosine phosphorylation is generally understood to govern many aspects of cellular behavior. The biological consequences of this signaling pathway are important because the levels of protein tyrosine phosphorylation are frequently elevated in cancer cells. In the classic paradigm, tyrosine kinases promote tumor cell growth, survival, and invasiveness, whereas tyrosine phosphatases negatively regulate these same behaviors. Here, we identify one particular tyrosine phosphatase, low molecular weight tyrosine phosphatase (LMW-PTP), which is frequently overexpressed in transformed cells. We also show that overexpression of LMW-PTP is sufficient to confer transformation upon non-transformed epithelial cells. Notably, we show that the EphA2 receptor tyrosine kinase is a prominent substrate for LMW-PTP and that the oncogenic activities of LMW-PTP result from altered EphA2 expression and function. These results suggest a role for LMW-PTP in transformation progression and link its oncogenic potential to EphA2.

  15. Identification of nitrated tyrosine residues of protein kinase G-Iα by mass spectrometry.

    Science.gov (United States)

    Lu, Jingshan; Yao, Ikuko; Shimojo, Masahito; Katano, Tayo; Uchida, Hitoshi; Setou, Mitsutoshi; Ito, Seiji

    2014-02-01

    The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-Iα expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-Iα, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-Iα; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-Iα activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 μM to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-Iα was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.

  16. Src kinases and ERK activate distinct responses to Stitcher receptor tyrosine kinase signaling during wound healing in Drosophila.

    Science.gov (United States)

    Tsarouhas, Vasilios; Yao, Liqun; Samakovlis, Christos

    2014-04-15

    Metazoans have evolved efficient mechanisms for epidermal repair and survival following injury. Several cellular responses and key signaling molecules that are involved in wound healing have been identified in Drosophila, but the coordination of cytoskeletal rearrangements and the activation of gene expression during barrier repair are poorly understood. The Ret-like receptor tyrosine kinase (RTK) Stitcher (Stit, also known as Cad96Ca) regulates both re-epithelialization and transcriptional activation by Grainy head (Grh) to induce restoration of the extracellular barrier. Here, we describe the immediate downstream effectors of Stit signaling in vivo. Drk (Downstream of receptor kinase) and Src family tyrosine kinases bind to the same docking site in the Stit intracellular domain. Drk is required for the full activation of transcriptional responses but is dispensable for re-epithelialization. By contrast, Src family kinases (SFKs) control both the assembly of a contractile actin ring at the wound periphery and Grh-dependent activation of barrier-repair genes. Our analysis identifies distinct pathways mediating injury responses and reveals an RTK-dependent activation mode for Src kinases and their central functions during epidermal wound healing in vivo.

  17. Identification and Targeting of Upstream Tyrosine Kinases Mediating PI3 Kinase Activation in PTEN Deficient Prostate Cancer

    Science.gov (United States)

    2011-06-01

    pAkt, phospho-Akt; Ab, antibody ; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; GPCR , G protein-coupled receptor...tyrosine phosphorylated proteins, but they were not recognized by an anti-pYxxM motif antibody and were not found in PTEN deficient PC3 PCa cells. LC/MS/MS...immunoblotted the p85 immunoprecipitates with a pYxxM motif specific antibody . This antibody weakly detected several discrete p85 associated proteins

  18. Functional interaction of caveolin-1 with Bruton's tyrosine kinase and Bmx.

    Science.gov (United States)

    Vargas, Leonardo; Nore, Beston F; Berglof, Anna; Heinonen, Juhana E; Mattsson, Pekka T; Smith, C I Edvard; Mohamed, Abdalla J

    2002-03-15

    Bruton's tyrosine kinase (Btk), a member of the Tec family of protein-tyrosine kinases, has been shown to be crucial for B cell development, differentiation, and signaling. Mutations in the Btk gene lead to X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice. Using a co-transfection approach, we present evidence here that Btk interacts physically with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of caveolae membranes. In addition, we found that native Bmx, another member of the Tec family kinases, is associated with endogenous caveolin-1 in primary human umbilical vein endothelial cells. Second, in transient transfection assays, expression of caveolin-1 leads to a substantial reduction in the in vivo tyrosine phosphorylation of both Btk and its constitutively active form, E41K. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82--101) functionally suppressed the autokinase activity of purified recombinant Btk protein. Third, we demonstrate that mouse splenic B-lymphocytes express substantial amounts of caveolin-1. Interestingly, caveolin-1 was found to be constitutively phosphorylated on tyrosine 14 in these cells. The expression of caveolin-1 in B-lymphocytes and its interaction with Btk may have implications not only for B cell activation and signaling, but also for antigen presentation.

  19. Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization.

    Science.gov (United States)

    Mohanty, Smita; Oruganty, Krishnadev; Kwon, Annie; Byrne, Dominic P; Ferries, Samantha; Ruan, Zheng; Hanold, Laura E; Katiyar, Samiksha; Kennedy, Eileen J; Eyers, Patrick A; Kannan, Natarajan

    2016-02-01

    Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they

  20. Review of current classification, molecular alterations, and tyrosine kinase inhibitor therapies in myeloproliferative disorders with hypereosinophilia

    Directory of Open Access Journals (Sweden)

    Havelange V

    2013-08-01

    Full Text Available Violaine Havelange,1,2 Jean-Baptiste Demoulin1 1de Duve Institute, Université catholique de Louvain, Brussels, Belgium; 2Department of Hematology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium Abstract: Recent advances in our understanding of the molecular mechanisms underlying hypereosinophilia have led to the development of a 'molecular' classification of myeloproliferative disorders with eosinophilia. The revised 2008 World Health Organization classification of myeloid neoplasms included a new category called “myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1.” Despite the molecular heterogeneity of PDGFR (platelet-derived growth factor receptor rearrangements, tyrosine kinase inhibitors at low dose induce rapid and complete hematological remission in the majority of these patients. Other kinase inhibitors are promising. Further discoveries of new molecular alterations will direct the development of new specific inhibitors. In this review, an update of the classifications of myeloproliferative disorders associated with hypereosinophilia is discussed together with open and controversial questions. Molecular mechanisms and promising results of tyrosine kinase inhibitor treatments are reviewed. Keywords: hypereosinophilia, classification, myeloproliferative disorders, molecular alterations, tyrosine kinase inhibitor

  1. Neuroblastoma tyrosine kinase signaling networks involve FYN and LYN in endosomes and lipid rafts.

    Directory of Open Access Journals (Sweden)

    Juan Palacios-Moreno

    2015-04-01

    Full Text Available Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.

  2. Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain

    Science.gov (United States)

    In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct from one another, and thus while most animal receptor kinases are tyrosin...

  3. Tyrosine Kinase Inhibitors and Vascular Toxicity: Impetus for a Classification System?

    Science.gov (United States)

    Herrmann, Joerg

    2016-06-01

    The introduction of molecularly targeted therapies with tyrosine kinase inhibitors has revolutionized cancer therapy and has contributed to a steady decline in cancer-related mortality since the late 1990s. However, not only cardiac but also vascular toxicity has been reported for these agents, some as expected on-target effects (e.g., VEGF receptor inhibitors) and others as unanticipated events (e.g., BCR-Abl inhibitors). A sound understanding of these cardiovascular toxic effects is critical to advance mechanistic insight into vascular disease and clinical care. From a conceptual standpoint, there might be value in defining type I (permanent) and type II (transient) vascular toxicity. This review will focus on the tyrosine kinase inhibitors in current clinical use and their associated vascular side effects.

  4. Loss of Bmx nonreceptor tyrosine kinase prevents pressure overload-induced cardiac hypertrophy.

    Science.gov (United States)

    Mitchell-Jordan, Scherise A; Holopainen, Tanja; Ren, Shuxun; Wang, Sujing; Warburton, Sarah; Zhang, Michael J; Alitalo, Kari; Wang, Yibin; Vondriska, Thomas M

    2008-12-05

    Bmx nonreceptor tyrosine kinase has an established role in endothelial and lymphocyte signaling; however, its role in the heart is unknown. To determine whether Bmx participates in cardiac growth, we subjected mice deficient in the molecule (Bmx knockout mice) to transverse aortic constriction (TAC). In comparison with wild-type mice, which progressively developed massive hypertrophy following TAC, Bmx knockout mice were resistant to TAC-induced cardiac growth at the organ and cell level. Loss of Bmx preserved cardiac ejection fraction and decreased mortality following TAC. These findings are the first to demonstrate a necessary role for the Tec family of tyrosine kinases in the heart and reveal a novel regulator (Bmx) of pressure overload-induced hypertrophic growth.

  5. How tyrosine kinase inhibitors impair metabolism and endocrine system function: a systematic updated review.

    Science.gov (United States)

    Breccia, Massimo; Molica, Matteo; Alimena, Giuliana

    2014-12-01

    Tyrosine kinase inhibitors (TKIs) advent has deeply changed the outcome of chronic myeloid leukemia (CML) patients, with improved rates of response and overall survival. However, for this success some patients paid the price of a number of peculiar side effects, the so-called off-target side effects, specific for each one TKI. These effects are due to non-selective inhibition of other tyrosine kinase receptors, such as PDGFR, c-KIT, Src, VEGF. Consequences of this inhibition, some metabolic changes during the treatment with TKIs are reported. Aim of present review is to report metabolic changes and potential mechanisms involved in the pathogenesis related to imatinib, second (nilotinib and dasatinib) and third generation (bosutinib and ponatinib) TKIs.

  6. HER2-targeted therapy in breast cancer. Monoclonal antibodies and tyrosine kinase inhibitors

    DEFF Research Database (Denmark)

    Nielsen, Dorte Lisbet; Andersson, Michael; Kamby, Claus

    2008-01-01

    There is strong clinical evidence that trastuzumab, a monoclonal antibody targeting the human epidermal growth factor receptor (HER) two tyrosine kinase receptor, is an important component of first-line treatment of patients with HER2-positive metastatic breast cancer. In particular the combination...... with taxanes and vinorelbine has been established. In the preoperative setting inclusion of trastuzumab has significantly increased the pathological complete response rate. Results from large phase III trials evaluating adjuvant therapy in HER2-positive early breast cancer indicate that the addition...... of trastuzumab to chemotherapy improves disease-free and overall survival. The use of lapatinib, a dual tyrosine kinase inhibitor of both HER1 and HER2, in combination with capecitabine in the second-line treatment of HER2-positive patients with metastatic breast cancer previously treated with trastuzumab has...

  7. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E;

    1996-01-01

    area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane......The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  8. Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity.

    Science.gov (United States)

    Lee, S K; Su, B; Maher, S E; Bothwell, A L

    1994-05-01

    To characterize the function of the Ly-6A antigen in T cell activation, antisense Ly-6 RNA was expressed in a stably transfected antigen-specific T cell clone. Reduced Ly-6A expression results in inhibition of responses to antigen, anti-TCR (anti-T cell receptor) crosslinking and concanavalin A plus recombinant interleukin 1 and causes impairment of in vitro fyn tyrosine kinase activity. More substantial reduction of Ly-6A results in reduction of TCR expression. Analysis of mRNA species indicates that the reduction is specific for the TCR beta chain. These data demonstrate that Ly-6A may regulate TCR expression and may be involved in early events of T cell activation via regulation of fyn tyrosine kinase activity.

  9. Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicity of breast cancer cells.

    Science.gov (United States)

    Bagheri-Yarmand, R; Mandal, M; Taludker, A H; Wang, R A; Vadlamudi, R K; Kung, H J; Kumar, R

    2001-08-03

    Etk/Bmx, a member of the Tec family of nonreceptor protein-tyrosine kinases, is characterized by an N-terminal pleckstrin homology domain and has been shown to be a downstream effector of phosphatidylinositol 3-kinase. P21-activated kinase 1 (Pak1), another well characterized effector of phosphatidylinositol 3-kinase, has been implicated in the progression of breast cancer cells. In this study, we characterized the role of Etk in mammary development and tumorigenesis and explored the functional interactions between Etk and Pak1. We report that Etk expression is developmentally regulated in the mammary gland. Using transient transfection, coimmunoprecipitation and glutathione S-transferase-pull down assays, we showed that Etk directly associates with Pak1 via its N-terminal pleckstrin homology domain and also phosphorylates Pak1 on tyrosine residues. The expression of wild-type Etk in a non-invasive human breast cancer MCF-7 cells significantly increased proliferation and anchorage-independent growth of epithelial cancer cells. Conversely, expression of kinase-inactive mutant Etk-KQ suppressed the proliferation, anchorage-independent growth, and tumorigenicity of human breast cancer MDA-MB435 cells. These results indicate that Pak1 is a target of Etk and that Etk controls the proliferation as well as the anchorage-independent and tumorigenic growth of mammary epithelial cancer cells.

  10. Abl tyrosine kinases modulate cadherin-dependent adhesion upstream and downstream of Rho family GTPases.

    Science.gov (United States)

    Zandy, Nicole L; Pendergast, Ann Marie

    2008-02-15

    Formation and dissolution of intercellular adhesions are processes of paramount importance during tissue morphogenesis and for pathological conditions such as tumor metastasis. Cadherin-mediated intercellular adhesion requires dynamic regulation of the actin cytoskeleton. The pathways that link cadherin signaling to cytoskeletal regulation remain poorly defined. We have recently uncovered a novel role for the Abl family of tyrosine kinases linking cadherin-mediated adhesion to actin dynamics via the regulation of Rho family GTPases. Abl kinases are activated by cadherin engagement, localize to cell-cell junctions and are required for the formation of adherens junctions. Notably, we showed that Abl kinases are required for Rac activation during formation of adherens junctions, and also regulate a Rho-ROCK-myosin signaling pathway that is required for the maintenance of intercellular adhesion. Here we show that Abl kinases regulate the formation and strengthening of adherens junctions downstream of active Rac, and that Abl tyrosine kinases are components of a positive feed-back loop that employs the Crk/CrkL adaptor proteins to promote the formation and maturation of adherens junctions.

  11. Loss of Bmx Non-Receptor Tyrosine Kinase Prevents Pressure Overload-Induced Cardiac Hypertrophy

    OpenAIRE

    2008-01-01

    Bmx non-receptor tyrosine kinase has an established role in endothelial and lymphocyte signaling, however its role in the heart is unknown. To determine whether Bmx participates in cardiac growth, we subjected mice deficient in the molecule (Bmx KO mice) to transverse aortic constriction (TAC). In comparison to WT mice, which progressively developed massive hypertrophy following TAC, Bmx KO mice were resistant to TAC-induced cardiac growth at the organ and cell level. Loss of Bmx preserved ca...

  12. Bmx Tyrosine Kinase Transgene Induces Skin Hyperplasia, Inflammatory Angiogenesis, and Accelerated Wound Healing

    OpenAIRE

    2004-01-01

    The Bmx gene, a member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in arterial endothelium and in certain hematopoietic and epithelial cells. Previous in vitro studies have implicated Bmx signaling in cell migration and survival and suggested that it contributes to the progression of prostate carcinomas. However, the function of Bmx in normal tissues in vivo is unknown. We show here that Bmx expression is induced in skin keratinocytes during wound healing. To analy...

  13. Receptor tyrosine kinase signaling regulates replication of the peste des petits ruminants virus.

    Science.gov (United States)

    Chaudhary, K; Chaubey, K K; Singh, S V; Kumar, N

    2015-03-01

    In this study, we found out that blocking the receptor tyrosine kinase (RTK) signaling in Vero cells by tryphostin AG879 impairs the in vitro replication of the peste des petits ruminants virus (PPRV). A reduced virus replication in Trk1-knockdown (siRNA) Vero cells confirmed the essential role of RTK in the virus replication, in particular a specific regulation of viral RNA synthesis. These data represent the first evidence that the RTK signaling regulates replication of a morbillivirus.

  14. Cross-membrane signal transduction of receptor tyrosine kinases (RTKs): from systems biology to systems pharmacology.

    Science.gov (United States)

    Benson, Neil; van der Graaf, Piet H; Peletier, Lambertus A

    2013-03-01

    Receptor tyrosine kinases are high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. They straddle the cell wall and play an important role in cross-membrane signalling. We present a two-component systems pharmacology model based on the local physiology and identify characteristic features of its dynamics. We thus present a transparent tool for studying the effects of drug intervention and ways of administration on cross-membrane signalling through these receptors.

  15. Functions of the Lyn tyrosine kinase in health and disease

    Directory of Open Access Journals (Sweden)

    Ingley Evan

    2012-07-01

    Full Text Available Abstract Src family kinases such as Lyn are important signaling intermediaries, relaying and modulating different inputs to regulate various outputs, such as proliferation, differentiation, apoptosis, migration and metabolism. Intriguingly, Lyn can mediate both positive and negative signaling processes within the same or different cellular contexts. This duality is exemplified by the B-cell defect in Lyn−/− mice in which Lyn is essential for negative regulation of the B-cell receptor; conversely, B-cells expressing a dominant active mutant of Lyn (Lynup/up have elevated activities of positive regulators of the B-cell receptor due to this hyperactive kinase. Lyn has well-established functions in most haematopoietic cells, viz. progenitors via influencing c-kit signaling, through to mature cell receptor/integrin signaling, e.g. erythrocytes, platelets, mast cells and macrophages. Consequently, there is an important role for this kinase in regulating hematopoietic abnormalities. Lyn is an important regulator of autoimmune diseases such as asthma and psoriasis, due to its profound ability to influence immune cell signaling. Lyn has also been found to be important for maintaining the leukemic phenotype of many different liquid cancers including acute myeloid leukaemia (AML, chronic myeloid leukaemia (CML and B-cell lymphocytic leukaemia (BCLL. Lyn is also expressed in some solid tumors and here too it is establishing itself as a potential therapeutic target for prostate, glioblastoma, colon and more aggressive subtypes of breast cancer. Lay Abstract To relay information, a cell uses enzymes that put molecular markers on specific proteins so they interact with other proteins or move to specific parts of the cell to have particular functions. A protein called Lyn is one of these enzymes that regulate information transfer within cells to modulate cell growth, survival and movement. Depending on which type of cell and the source of the information

  16. Overexpression of the potential kinase serine/ threonine/tyrosine kinase 1 (STYK 1) in castration-resistant prostate cancer.

    Science.gov (United States)

    Chung, Suyoun; Tamura, Kenji; Furihata, Mutsuo; Uemura, Motohide; Daigo, Yataro; Nasu, Yasutomo; Miki, Tsuneharu; Shuin, Taro; Fujioka, Tomoaki; Nakamura, Yusuke; Nakagawa, Hidewaki

    2009-11-01

    Despite high response rates and clinical benefits, androgen ablation often fails to cure advanced or relapsed prostate cancer because castration-resistant prostate cancer (CRPC) cells inevitably emerge. CRPC cells not only grow under castration, but also behave more aggressively, indicating that a number of malignant signaling pathways are activated in CRPC cells as well as androgen receptor signaling. Based on information from the gene expression profiles of clinical CRPC cells, we here identified one overexpressed gene, serine/threonine/tyrosine kinase 1 (STYK1), encoding a potential kinase, as a molecular target for CRPC. RNA and immunohistochemical analyses validated the overexpression of STYK1 in prostate cancer cells, and its expression was distinct in CRPC cells. Knockdown of STYK1 by siRNA resulted in drastic suppression of prostate cancer cell growth and, concordantly, enforced expression of STYK1 promoted cell proliferation, whereas ectopic expression of a kinase-dead mutant STYK1 did not. An in vitro kinase assay using recombinant STYK1 demonstrated that STYK1 could have some potential as a kinase, although its specific substrates are unknown. These findings suggest that STYK1 could be a possible molecular target for CRPC, and small molecules specifically inhibiting STYK1 kinase could be a possible approach for the development of novel CRPC therapies.

  17. BMX tyrosine kinase gene is expressed in granulocytes and myeloid leukaemias.

    Science.gov (United States)

    Kaukonen, J; Lahtinen, I; Laine, S; Alitalo, K; Palotie, A

    1996-09-01

    The growth and maturation of haemopoietic cells is regulated by signal transduction through tyrosine protein kinases. Recently, a novel cytoplasmic tyrosine kinase gene in chromosome X, called Bmx, was identified in human bone marrow RNA. Bmx belongs to a subfamily of tyrosine kinases which are expressed in various haemopoietic cell lineages. We studied Bmx expression using RT-PCR of RNA from fractionated peripheral blood leucocytes, progenitor-enriched fractions of cord blood and from bone marrow or peripheral blood samples from leukaemia patients. Bmx was strongly expressed in haemopoietic tissues and enhanced in neutrophilic granulocytes. Bmx mRNA was also found in CD34-positive progenitor cells from cord blood. All samples (10/10) of patients with acute myeloid leukaemia and (4/4) with chronic myeloid leukaemia showed expression of Bmx. In contrast, none of the samples of acute lymphoid leukaemia (0/8) and only one out of six samples of chronic lymphoid leukaemia expressed Bmx. In conclusion, Bmx expression seems to be associated with myelopoiesis.

  18. A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control.

    Science.gov (United States)

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Thon, Lutz; Mamat, Uwe; Bellosta, Paola; Basilico, Claudio; Adam, Dieter; Paus, Ralf; Bulfone-Paus, Silvia

    2005-12-21

    Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a 'promiscuous liaison' between both receptors that enables interleukin (IL)-15 to transactivate the signaling pathway of a tyrosine kinase. IL-15 protects murine L929 fibroblasts from tumor necrosis factor alpha (TNFalpha)-induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl-overexpressing fibroblasts are TNFalpha-resistant. IL-15Ralpha and Axl colocalize on the cell membrane and co-immunoprecipitate even in the absence of IL-15, whereby the extracellular part of Axl proved to be essential for Axl/IL-15Ralpha interaction. Most strikingly, IL-15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL-15Ralpha, and activation of the phosphatidylinositol 3-kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild-type but not Axl-/- or IL-15Ralpha-/- mice. Thus, IL-15-induced protection from TNFalpha-mediated cell death involves a hitherto unknown IL-15 receptor complex, consisting of IL-15Ralpha and Axl RTK, and requires their reciprocal activation initiated by ligand-induced IL-15Ralpha.

  19. Treatment of Recurrent Intracranial Hemangiopericytoma with SRC-Related Tyrosine Kinase Targeted Therapy: A Case Report

    Directory of Open Access Journals (Sweden)

    Katherine B. Peters

    2010-04-01

    Full Text Available Hemangiopericytoma (HPC is a rare sarcomatous tumor arising from pericytes, a support cell found in blood vessels. These tumors can occur throughout the body, particularly in the lower extremities and retroperitoneum. In rare circumstances, HPCs can arise from the meninges. In these cases, they behave similar to meningiomas, in particular angiomatous meningiomas, but tend to be more aggressive and are likely to recur. Treatment usually focuses on surgical resection and radiotherapy with possible inclusion of chemotherapy for control of recurrent disease. We describe a case of recurrent right temporal HPC that first manifested as a paraneoplastic syndrome of oncogenic osteomalacia. Despite maximum therapy, this patient experienced multiple recurrences of the tumor, and immunohistochemical analysis revealed overexpression of platelet-derived growth factor receptor, a member of the SRC-related tyrosine kinases. After multiple recurrences, the patient’s tumor has been stable with treatment with monotherapy utilizing molecularly targeted therapy to SRC-related tyrosine kinases. This is the first case report of the treatment of recurrent meningeal HPC with molecularly targeted therapy to SRC-related tyrosine kinases.

  20. Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases

    DEFF Research Database (Denmark)

    Pandey, A.; Blagoev, B.; Kratchmarova, I.;

    2002-01-01

    We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling mo...

  1. MHC class I signaling in T cells leads to tyrosine kinase activity and PLC-gamma 1 phosphorylation

    DEFF Research Database (Denmark)

    Skov, S; Odum, Niels; Claesson, M H

    1995-01-01

    phosphorylation and the subsequent calcium response. The early tyrosine kinase activity was found to be dependent on expression of the TCR/CD3 complex and the CD45 molecule on the surface of the T cells. Furthermore, MHC-I cross-linking was shown to tyrosine phosphorylate PLC-gamma 1 (phospholipase C-gamma 1...

  2. Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain

    Directory of Open Access Journals (Sweden)

    Man-Ho eOh

    2012-08-01

    Full Text Available In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct and thus while most animal receptor kinases are tyrosine kinases the plant receptor kinases are classified as serine/threonine kinases. One of the best studied plant receptor kinases is BRASSINOSTEROID INSENSITIVE 1 (BRI1, which functions in brassinosteroid (BR signaling. Consistent with its classification, BRI1 was shown in early studies to autophosphorylate in vitro exclusively on serine and threonine residues and subsequently numerous specific phosphoserine and phosphothreonine sites were identified. However, several sites of tyrosine autophosphorylation have recently been identified establishing that BRI1 is a dual-specificity kinase. This raises the paradox that BRI1 contains phosphotyrosine but was only observed to autophosphorylate on serine and threonine sites. In the present study, we demonstrate that autophosphorylation on threonine and tyrosine (and presumably serine residues is a post-translational modification, ruling out a co-translational mechanism that could explain the paradox. Moreover, we show that in general, autophosphorylation of the recombinant protein appears to be hierarchal and proceeds in the order: phosphoserine > phosphothreonine > phosphotyrosine. This may explain why tyrosine autophosphorylation was not observed in some studies. Finally, we also show that the juxtamembrane domain of BRI1 is an activator of the kinase domain, and that kinase specificity (serine/threonine versus tyrosine can be affected by residues outside of the kinase domain. This may have implications for identification of signature motifs that distinguish serine/threonine kinases from dual-specificity kinases.

  3. Negative Regulation of Receptor Tyrosine Kinase (RTK Signaling: A Developing Field

    Directory of Open Access Journals (Sweden)

    Fernanda Ledda

    2007-01-01

    Full Text Available ophic factors control cellular physiology by activating specific receptor tyrosine kinases (RTKs. While the over activation of RTK signaling pathways is associated with cell growth and cancer, recent findings support the concept that impaired down-regulation or deactivation of RTKs may also be a mechanism involved in tumor formation. Under this perspective, the molecular determinants of RTK signaling inhibition may act as tumor-suppressor genes and have a potential role as tumor markers to monitor and predict disease progression. Here, we review the current understanding of the physiological mechanisms that attenuate RTK signaling and discuss evidence that implicates deregulation of these events in cancer.Abbreviations: BDP1: Brain-derived phosphatase 1; Cbl: Casitas B-lineage lymphoma; CIN-85: Cbl-interacting protein of 85 kDa; DER: Drosophila EGFR; EGFR: Epidermal growth factor receptor; ERK 1/2: Extracellular signal-regulated kinase 1/2; Grb2: Growth factor receptor-bound protein 2; HER2: Human epidermal growth factor receptor 2; LRIG: Leucine-rich repeats and immunoglobulin-like domain 1; MAPK: Mitogen-activated protein kinase; Mig 6: Mitogen-inducible gene 6; PTEN: Phosphatase and tensin homologue; RET: Rearranged in transformation; RTK: Receptor tyrosine kinase. SH2 domain: Src-homology 2 domain; SH3 domain: Src-homology 3 domain; Spry: Sprouty.

  4. NPM-ALK: The Prototypic Member of a Family of Oncogenic Fusion Tyrosine Kinases

    Directory of Open Access Journals (Sweden)

    Joel D. Pearson

    2012-01-01

    Full Text Available Anaplastic lymphoma kinase (ALK was first identified in 1994 with the discovery that the gene encoding for this kinase was involved in the t(2;5(p23;q35 chromosomal translocation observed in a subset of anaplastic large cell lymphoma (ALCL. The NPM-ALK fusion protein generated by this translocation is a constitutively active tyrosine kinase, and much research has focused on characterizing the signalling pathways and cellular activities this oncoprotein regulates in ALCL. We now know about the existence of nearly 20 distinct ALK translocation partners, and the fusion proteins resulting from these translocations play a critical role in the pathogenesis of a variety of cancers including subsets of large B-cell lymphomas, nonsmall cell lung carcinomas, and inflammatory myofibroblastic tumours. Moreover, the inhibition of ALK has been shown to be an effective treatment strategy in some of these malignancies. In this paper we will highlight malignancies where ALK translocations have been identified and discuss why ALK fusion proteins are constitutively active tyrosine kinases. Finally, using ALCL as an example, we will examine three key signalling pathways activated by NPM-ALK that contribute to proliferation and survival in ALCL.

  5. Spleen tyrosine kinase inhibitors for rheumatoid arthritis: where are we now?

    Science.gov (United States)

    Scott, Ian C; Scott, David L

    2014-03-01

    The development of small-molecule inhibitors of inflammatory cascade signaling kinases offers a potential approach to treating rheumatoid arthritis (RA). Spleen tyrosine kinase is one such tyrosine kinase. Recent research efforts have focussed on the development and testing of a spleen tyrosine kinase inhibitor, fostamatinib. We reviewed the results of the clinical trials of fostamatinib in RA with the aim of outlining its clinical efficacy and the nature and frequency of its main adverse events. To date, this drug has been evaluated in over 3,200 RA patients enrolled in three phase II, one phase IIb and three phase III trials. These studies showed fostamatinib was effective. In four trials in which patients received 100 mg twice daily, fostamatinib reduced inflammatory synovitis; the relative risks of achieving American College of Rheumatology Responder rates compared with placebo in the combined studies ranged from 1.6 for 20 % of responders to 3.7 for 70 % of responders. There was a similar relative risk of achieving a clinically meaningful reduction in disability of 1.6 for the chance of patients achieving a reduction in health assessment questionnaire scores of 0.22 or more. Three of the trials examined the impact of fostamatinib on erosive radiographic damage using changes in the modified total Sharp score. None of them provided any evidence for a significant effect of fostamatinib on erosive damage over 6 months. All the trials included descriptions of adverse events. Hypertension was common, involving over 40 % of patients treated. Other common adverse events included diarrhoea, neutropenia and increases in hepatic enzyme levels. Some patients developed infections. On the conclusion of the phase III trials, one of the main pharmaceutical sponsors decided not to further develop fostamatinib for RA.

  6. Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39.

    Science.gov (United States)

    Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

    2016-04-22

    RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39.

  7. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Science.gov (United States)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

  8. Hierarchical Disabled-1 Tyrosine Phosphorylation in Src family Kinase Activation and Neurite Formation

    Science.gov (United States)

    Katyal, Sachin; Gao, Zhihua; Monckton, Elizabeth; Glubrecht, Darryl; Godbout, Roseline

    2013-01-01

    There are two developmentally regulated alternatively spliced forms of Disabled-1 (Dab1) in the chick retina: an early form (Dab1-E) expressed in retinal precursor cells and a late form (Dab1-L) expressed in neuronal cells. The main difference between these two isoforms is the absence of two Src family kinase (SFK) recognition sites in Dab1-E. Both forms retain two Abl/Crk/Nck recognition sites implicated in the recruitment of SH2 domain-containing signaling proteins. One of the Dab1-L-specific SFK recognition sites, at tyrosine(Y)-198, has been shown to be phosphorylated in Reelin-stimulated neurons. Here, we use Reelin-expressing primary retinal cultures to investigate the role of the four Dab1 tyrosine phosphorylation sites on overall tyrosine phosphorylation, Dab1 phosphorylation, SFK activation and neurite formation. We show that Y198 is essential but not sufficient for maximal Dab1 phosphorylation, SFK activation and neurite formation, with Y232 and Y220 playing particularly important roles in SFK activation and neuritogenesis, and Y185 having modifying effects secondary to Y232 and Y220. Our data support a role for all four Dab1 tyrosine phosphorylation sites in mediating the spectrum of activities associated with Reelin-Dab1 signaling in neurons. PMID:17350651

  9. Autophosphorylation of the Bacterial Tyrosine-Kinase CpsD Connects Capsule Synthesis with the Cell Cycle in Streptococcus pneumoniae

    NARCIS (Netherlands)

    Nourikyan, Julien; Kjos, Morten; Mercy, Chryslène; Cluzel, Caroline; Morlot, Cécile; Noirot-Gros, Marie-Francoise; Guiral, Sébastien; Lavergne, Jean-Pierre; Veening, Jan-Willem; Grangeasse, Christophe

    2015-01-01

    Bacterial capsular polysaccharides (CPS) are produced by a multi-protein membrane complex, in which a particular type of tyrosine-autokinases named BY-kinases, regulate their polymerization and export. However, our understanding of the role of BY-kinases in these processes remains incomplete. In the

  10. Interleukin 7 receptor functions by recruiting the tyrosine kinase p59fyn through a segment of its cytoplasmic tail.

    OpenAIRE

    1992-01-01

    Engagement of the cell surface receptor for interleukin 7 (IL-7R) provokes protein tyrosine phosphorylation, although the receptor lacks a kinase catalytic domain in its cytoplasmic tail. The molecular basis of this response is not known. Here we report that the IL-7R functions by recruiting p59fyn, an intracellular tyrosine kinase of the src family. Treatment of pre-B cells with IL-7 causes an enhancement of the catalytic activity of p59fyn, but not of the related kinase p62yes. IL-7-depende...

  11. Multiple Functions of Let-23, a Caenorhabditis Elegans Receptor Tyrosine Kinase Gene Required for Vulval Induction

    Science.gov (United States)

    Aroian, R. V.; Sternberg, P. W.

    1991-01-01

    The let-23 gene, which encodes a putative tyrosine kinase of the epidermal growth factor (EGF) receptor subfamily, has multiple functions during Caenorhabditis elegans development. We show that let-23 function is required for vulval precursor cells (VPCs) to respond to the signal that induces vulval differentiation: a complete loss of let-23 function results in no induction. However, some let-23 mutations that genetically reduce but do not eliminate let-23 function result in VPCs apparently hypersensitive to inductive signal: as many as five of six VPCs can adopt vulval fates, in contrast to the three that normally do. These results suggest that the let-23 receptor tyrosine kinase controls two opposing pathways, one that stimulates vulval differentiation and another that negatively regulates vulval differentiation. Furthermore, analysis of 16 new let-23 mutations indicates that the let-23 kinase functions in at least five tissues. Since various let-23 mutant phenotypes can be obtained independently, the let-23 gene is likely to have tissue-specific functions. PMID:2071015

  12. Erkitinib, a novel EGFR tyrosine kinase inhibitor screened using a ProteoChip system from a phytochemical library

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eung-Yoon; Choi, Young-Jin [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of); Park, Chan-Won [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Kang, In-Cheol, E-mail: ickang@hoseo.edu [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of)

    2009-11-20

    Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer. Therefore PTK inhibitors are currently under intensive investigation as potential drug candidates. Herein, we report on a ProteoChip-based screening of an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, Erkitinibs, from phytochemical libraries. PLC-{gamma}-1 was used as a substrate immobilized on a ProteoChip and incubated with an EGFR kinase to phosphorylate tyrosine residues of the substrate, followed by a fluorescence detection of the substrate recognized by a phospho-specific monoclonal antibody. Erkitinibs inhibited HeLa cell proliferation in a dose-dependent manner. In conclusion, these data suggest that Erkitinibs can be a specific inhibitor of an EGFR kinase and can be further developed as a potent anti-tumor agent.

  13. Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors.

    Science.gov (United States)

    Obata, Y; Horikawa, K; Takahashi, T; Akieda, Y; Tsujimoto, M; Fletcher, J A; Esumi, H; Nishida, T; Abe, R

    2017-02-13

    Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek-Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)'s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs.Oncogene advance online publication, 13 February 2017; doi:10.1038/onc.2016.519.

  14. Application of computational approaches to study signalling networks of nuclear and Tyrosine kinase receptors

    Directory of Open Access Journals (Sweden)

    Rebaï Ahmed

    2010-10-01

    Full Text Available Abstract Background Nuclear receptors (NRs and Receptor tyrosine kinases (RTKs are essential proteins in many cellular processes and sequence variations in their genes have been reported to be involved in many diseases including cancer. Although crosstalk between RTK and NR signalling and their contribution to the development of endocrine regulated cancers have been areas of intense investigation, the direct coupling of their signalling pathways remains elusive. In our understanding of the role and function of nuclear receptors on the cell membrane the interactions between nuclear receptors and tyrosine kinase receptors deserve further attention. Results We constructed a human signalling network containing nuclear receptors and tyrosine kinase receptors that identified a network topology involving eleven highly connected hubs. We further developed an integrated knowledge database, denominated NR-RTK database dedicated to human RTKs and NRs and their vertebrate orthologs and their interactions. These interactions were inferred using computational tools and those supported by literature evidence are indicated. NR-RTK database contains links to other relevant resources and includes data on receptor ligands. It aims to provide a comprehensive interaction map that identifies complex dynamics and potential crosstalk involved. Availability: NR-RTK database is accessible at http://www.bioinfo-cbs.org/NR-RTK/ Conclusions We infer that the NR-RTK interaction network is scale-free topology. We also uncovered the key receptors mediating the signal transduction between these two types of receptors. Furthermore, NR-RTK database is expected to be useful for researchers working on various aspects of the molecular basis of signal transduction by RTKs and NRs. Reviewers This article was reviewed by Professor Paul Harrison (nominated by Dr. Mark Gerstein, Dr. Arcady Mushegian and Dr. Anthony Almudevar.

  15. Complement receptor-3 negatively regulates the phagocytosis of degenerated myelin through tyrosine kinase Syk and cofilin

    Directory of Open Access Journals (Sweden)

    Hadas Smadar

    2012-07-01

    Full Text Available Abstract Background Intact myelin, which normally surrounds axons, breaks down in Wallerian degeneration following axonal injury and during neurodegenerative diseases such as multiple sclerosis. Clearance of degenerated myelin by phagocytosis is essential since myelin impedes repair and exacerbates damage. CR3 (complement receptor-3 is a principal phagocytic receptor in myelin phagocytosis. We studied how tyrosine kinase Syk (spleen tyrosine kinase and cofilin control phagocytosis of degenerated myelin by CR3 in microglia and macrophages. Syk is a non-receptor tyrosine kinase that CR3 recruits to convey cellular functions. Cofilin is an actin-depolymerizing protein that controls F-actin (filamentous actin remodeling (i.e., disassembly and reassembly by shifting between active unphosphorylated and inactive phosphorylated states. Results Syk was continuously activated during prolonged phagocytosis. Phagocytosis increased when Syk activity and expression were reduced, suggesting that normally Syk down regulates CR3-mediated myelin phagocytosis. Levels of inactive p-cofilin (phosphorylated cofilin decreased transiently during prolonged phagocytosis. In contrast, p-cofilin levels decreased continuously when Syk activity and expression were continuously reduced, suggesting that normally Syk advances the inactive state of cofilin. Observations also revealed inverse relationships between levels of phagocytosis and levels of inactive p-cofilin, suggesting that active unphosphorylated cofilin advances phagocytosis. Active cofilin could advance phagocytosis by promoting F-actin remodeling, which supports the production of membrane protrusions (e.g., filopodia, which, as we also revealed, are instrumental in myelin phagocytosis. Conclusions CR3 both activates and downregulates myelin phagocytosis at the same time. Activation was previously documented. We presently demonstrate that downregulation is mediated through Syk, which advances the inactive

  16. Identification Of Akn-032, A Novel 2-Aminopyrazine Tyrosine Kinase Inhibitor, With Significant Preclinical Activity In Acute Myeloid Leukemia

    OpenAIRE

    Eriksson, Anna; Höglund, Martin; Lindhagen, Elin; Åleskog, Anna; Hassan, Sadia Bashir; Ekholm, Carina; Fhölenhag, Karin; Jensen, Annika Jenmalm; Löthgren, Agneta; Scobie, Martin; Larsson, Rolf; Parrow, Vendela

    2010-01-01

    Abstract Aberrant signal transduction by mutant or overexpressed protein kinases has emerged as a promising target for treatment of acute myeloid leukemia (AML). We here present a novel low molecular weight kinase inhibitor, AKN-032, targeting the FMS-like tyrosine kinase 3 (FLT3) and discovered in a new type of screening funnel combining the target therapy approach with sequential cellular screens. AKN-032 was identified among 150 selected hits from three different high throughput...

  17. A Src-like inactive conformation in the abl tyrosine kinase domain.

    Directory of Open Access Journals (Sweden)

    Nicholas M Levinson

    2006-05-01

    Full Text Available The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML. The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG motif is flipped by approximately 180 degrees with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP-peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR-Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain.

  18. Receptor Tyrosine Kinase EphB4 Is a Survival Factor in Breast Cancer

    OpenAIRE

    Kumar, S. Ram; Singh, Jasbir; Xia, Guangbin; Krasnoperov, Valery; Hassanieh, Loubna; Ley, Eric J.; Scehnet, Jeffrey; Kumar, Neil G.; Hawes, Debra; Press, Michael F.; Weaver, Fred A.; Gill, Parkash S.

    2006-01-01

    EphB4, a member of the largest family of receptor tyrosine kinases, is normally expressed on endothelial and neuronal cells. Although aberrant expression of EphB4 has been reported in several human tumors, including breast cancer, its functional significance is not understood. We report here that EphB4 is expressed in 7 of 12 (58%) human breast cancer specimens and 4 of 4 (100%) breast tumor cell lines examined. Overexpression of EphB4 in breast cancer cells was driven by gene amplification a...

  19. Efficacy of HER2-targeted therapy in metastatic breast cancer. Monoclonal antibodies and tyrosine kinase inhibitors

    DEFF Research Database (Denmark)

    Nielsen, Dorte L; Kümler, Iben; Palshof, Jesper Andreas;

    2013-01-01

    Therapies targeting the human epidermal growth factor receptor (HER) 2 are effective in metastatic breast cancer (MBC). We review the efficacy of HER2-directed therapies, focussing on monoclonal antibodies and tyrosine kinase inhibitors targeting HER2 that have been tested in phase II-III studies...... to those obtained for capecitabine plus lapatinib (48%), continuing trastuzumab in combination with capecitabine (48%), pertuzumab plus trastuzumab (24%), and neratinib (24%). Strategies combining multiple HER2-directed therapies might yield additive or synergistic effects and lead to improved outcome...

  20. Role of receptor tyrosine kinases in gastric cancer: New targets for a selective therapy

    Institute of Scientific and Technical Information of China (English)

    JC Becker; C Müller-Tidow; H Serve; W Domschke; T Pohle

    2006-01-01

    Receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor family participate in several steps of tumor formation including proliferation and metastatic spread. Several known RTKs are upregulated in gastric cancer being prime targets of a tailored therapy. Only preliminary data exist, however, on the use of the currently clinically available drugs such as trastuzumab,cetuximab, bevacizumab, gefitinib, erlotinib, and imatinib in the setting of gastric cancer. Preclinical data suggest a potential benefit of their use, especially in combination with "conventional" cytostatic therapy. This review summarizes the current knowledge about their use in cancer therapy as well as new approaches and drugs to optimize treatment success.

  1. Pregnancies in patients with chronic myeloid leukemia treated with tyrosine kinase inhibitor.

    Science.gov (United States)

    Zhou, Li; You, Jian-Hua; Wu, Wen; Li, Jun-Min; Shen, Zhi-Xiang; Wang, Ai-Hua

    2013-10-01

    We presented our experience in chronic myeloid leukemia (CML) patients who conceived children and/or became pregnant while receiving tyrosine kinase inhibitor (TKI). Among 7 male patients, 7 pregnancies resulted in the birth of 7 healthy babies. Among 18 female patients, 8 ended in elective abortion; 3 had spontaneous abortion, and 7 carried to term, resulting in the birth of 8 healthy babies. All children have normal growth and development. All patients remain in TKI therapy and in good response. It is suggested that female patients are advised to practice adequate contraception. No special precautions apply for male patients receiving TKI.

  2. Role of tyrosine-kinase inhibitors in myeloproliferative neoplasms: comparative lessons learned

    Directory of Open Access Journals (Sweden)

    Pinilla-Ibarz J

    2016-08-01

    Full Text Available Javier Pinilla-Ibarz, Kendra L Sweet, Gabriela M Corrales-Yepez, Rami S Komrokji Department of Malignant Hematology, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Abstract: An important pathogenetic distinction in the classification of myeloproliferative neoplasms (MPNs is the presence or absence of the BCR–ABL fusion gene, which encodes a unique oncogenic tyrosine kinase. The BCR–ABL fusion, caused by the formation of the Philadelphia chromosome (Ph through translocation, constitutes the disease-initiating event in chronic myeloid leukemia. The development of successive BCR–ABL-targeted tyrosine-kinase inhibitors has led to greatly improved outcomes in patients with chronic myeloid leukemia, including high rates of complete hematologic, cytogenetic, and molecular responses. Such levels of treatment success have long been elusive for patients with Ph-negative MPNs, because of the difficulties in identifying specific driver proteins suitable as drug targets. However, in recent years an improved understanding of the complex pathobiology of classic Ph-negative MPNs, characterized by variable, overlapping multimutation profiles, has prompted the development of better and more broadly targeted (to pathway rather than protein treatment options, particularly JAK inhibitors. In classic Ph-negative MPNs, overactivation of JAK-dependent signaling pathways is a central pathogenic mechanism, and mutually exclusive mutations in JAK2, MPL, and CALR linked to aberrant JAK activation are now recognized as key drivers of disease progression in myelofibrosis (MF. In clinical trials, the JAK1/JAK2 inhibitor ruxolitinib – the first therapy approved for MF worldwide – improved disease-related splenomegaly and symptoms independent of JAK2V617F mutational status, and prolonged survival compared with placebo or standard therapy in patients with advanced MF. In separate trials, ruxolitinib also provided comprehensive hematologic control in

  3. The Receptor Tyrosine Kinase Mertk Regulates Dendritic Cell Production of BAFF

    OpenAIRE

    Gohlke, P.R.; Williams, J. C.; Vilen, B J; Dillon, S.R.; Tisch, R; Matsushima, G.K.

    2009-01-01

    The MerTK receptor tyrosine kinase is an important negative regulator of dendritic cell function and is required to prevent B cell autoimmunity in vivo. It is not currently known however, if any causal relationship exists between these two aspects of MerTK function. We sought to determine if dendritic cells from mice lacking MerTK (mertk−/− mice) have characteristics that may aid in the development of B cell autoimmunity. Specifically, we found that mertk−/− mice contain an elevated number of...

  4. The TAM family: phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer.

    Science.gov (United States)

    Graham, Douglas K; DeRyckere, Deborah; Davies, Kurtis D; Earp, H Shelton

    2014-12-01

    The TYRO3, AXL (also known as UFO) and MERTK (TAM) family of receptor tyrosine kinases (RTKs) are aberrantly expressed in multiple haematological and epithelial malignancies. Rather than functioning as oncogenic drivers, their induction in tumour cells predominately promotes survival, chemoresistance and motility. The unique mode of maximal activation of this RTK family requires an extracellular lipid–protein complex. For example, the protein ligand, growth arrest-specific protein 6 (GAS6), binds to phosphatidylserine (PtdSer) that is externalized on apoptotic cell membranes, which activates MERTK on macrophages. This triggers engulfment of apoptotic material and subsequent anti-inflammatory macrophage polarization. In tumours, autocrine and paracrine ligands and apoptotic cells are abundant, which provide a survival signal to the tumour cell and favour an anti-inflammatory, immunosuppressive microenvironment. Thus, TAM kinase inhibition could stimulate antitumour immunity, reduce tumour cell survival, enhance chemosensitivity and diminish metastatic potential.

  5. Clinical pharmacology of tyrosine kinase inhibitors becoming generic drugs: the regulatory perspective.

    Science.gov (United States)

    Eckstein, Niels; Röper, Lea; Haas, Bodo; Potthast, Henrike; Hermes, Ulrike; Unkrig, Christoph; Naumann-Winter, Frauke; Enzmann, Harald

    2014-02-07

    Over the last decades, billions have been spent and huge efforts have been taken in basic and clinical cancer research [CA Cancer J Clin63:11-30]. About a decade ago, the arms race between drugs and cancer cells reached a new level by introduction of tyrosine kinase inhibitors (TKI) into pharmacological anti-cancer therapy. According to their molecular mechanism of action, TKI in contrast to so-called "classic" or "conventional" cytostatics belong to the group of targeted cancer medicines, characterized by accurately fitting with biological structures (i.e. active centers of kinases). Numerous (partly orphan) indications are covered by this new class of substances. Approximately ten years after the first substances of this class of medicines were authorized, patent protection will end within the next years. The following article covers clinical meaning and regulatory status of anti-cancer TKI and gives an outlook to what is expected from the introduction of generic anti-cancer TKI.

  6. The tyrosine kinase BMX is an essential mediator of inflammatory arthritis in a kinase-independent manner.

    Science.gov (United States)

    Gottar-Guillier, Marie; Dodeller, Francis; Huesken, Dieter; Iourgenko, Vadim; Mickanin, Craig; Labow, Mark; Gaveriaux, Samuel; Kinzel, Bernd; Mueller, Matthias; Alitalo, Kari; Littlewood-Evans, Amanda; Cenni, Bruno

    2011-05-15

    Inflammatory cytokines like TNF play a central role in autoimmune disorders such as rheumatoid arthritis. We identified the tyrosine kinase bone marrow kinase on chromosome X (BMX) as an essential component of a shared inflammatory signaling pathway. Transient depletion of BMX strongly reduced secretion of IL-8 in cell lines and primary human cells stimulated by TNF, IL-1β, or TLR agonists. BMX was required for phosphorylation of p38 MAPK and JNK, as well as activation of NF-κB. The following epistasis analysis indicated that BMX acts downstream of or at the same level as the complex TGF-β activated kinase 1 (TAK1)-TAK1 binding protein. At the cellular level, regulation of the IL-8 promoter required the pleckstrin homology domain of BMX, which could be replaced by an ectopic myristylation signal, indicating a requirement for BMX membrane association. In addition, activation of the IL-8 promoter by in vitro BMX overexpression required its catalytic activity. Genetic ablation of BMX conferred protection in the mouse arthritis model of passive K/BxN serum transfer, confirming that BMX is an essential mediator of inflammation in vivo. However, genetic replacement with a catalytically inactive BMX allele was not protective in the same arthritis animal model. We conclude that BMX is an essential component of inflammatory cytokine signaling and that catalytic, as well as noncatalytic functions of BMX are involved.

  7. Reduced Proteolytic Shedding of Receptor Tyrosine Kinases is a Post-Translational Mechanism of Kinase Inhibitor Resistance

    Science.gov (United States)

    Miller, Miles A.; Oudin, Madeleine J.; Sullivan, Ryan J.; Wang, Stephanie J.; Meyer, Aaron S.; Im, Hyungsoon; Frederick, Dennie T.; Tadros, Jenny; Griffith, Linda G.; Lee, Hakho; Weissleder, Ralph; Flaherty, Keith T.; Gertler, Frank B.; Lauffenburger, Douglas A.

    2016-01-01

    Kinase inhibitor resistance often involves upregulation of poorly understood “bypass” signaling pathways. Here, we show that extracellular proteomic adaptation is one path to bypass signaling and drug resistance. Proteolytic shedding of surface receptors, which can provide negative feedback on signaling activity, is blocked by kinase inhibitor treatment and enhances bypass signaling. In particular, MEK inhibition broadly decreases shedding of multiple receptor tyrosine kinases (RTKs) including HER4, MET, and most prominently AXL, an ADAM10 and ADAM17 substrate, thus increasing surface RTK levels and mitogenic signaling. Progression-free survival of melanoma patients treated with clinical BRAF/MEK inhibitors inversely correlates with RTK shedding reduction following treatment, as measured non-invasively in blood plasma. Disrupting protease inhibition by neutralizing TIMP1 improves MAPK inhibitor efficacy, and combined MAPK/AXL inhibition synergistically reduces tumor growth and metastasis in xenograft models. Altogether, extracellular proteomic rewiring through reduced RTK shedding represents a surprising mechanism for bypass signaling in cancer drug resistance. PMID:26984351

  8. Activation of nonreceptor tyrosine kinase Bmx/Etk mediated by phosphoinositide 3-kinase, epidermal growth factor receptor, and ErbB3 in prostate cancer cells.

    Science.gov (United States)

    Jiang, Xinnong; Borgesi, Robert A; McKnight, Nicole C; Kaur, Ramneet; Carpenter, Christopher L; Balk, Steven P

    2007-11-09

    Pathways activated downstream of constitutively active phosphatidylinositol (PI) 3-kinase in PTEN-deficient prostate cancer (PCa) cells are possible therapeutic targets. We found that the nonreceptor Tec family tyrosine kinase Bmx/Etk was activated by tyrosine phosphorylation downstream of Src and PI 3-kinase in PTEN-deficient LNCaP and PC3 PCa cells and that Bmx down-regulation by short interfering RNA markedly inhibited LNCaP cell growth. Bmx also associated with ErbB3 in LNCaP cells, and heregulin-beta1 enhanced this interaction and further stimulated Bmx activity. Epidermal growth factor (EGF) similarly stimulated an interaction between Bmx and EGF receptor and rapidly increased Bmx kinase activity. Bmx stimulation in response to heregulin-beta1 and EGF was Src-dependent, and heregulin-beta1 stimulation of Bmx was also PI 3-kinase-dependent. In contrast, the rapid tyrosine phosphorylation and activation of Bmx in response to EGF was PI 3-kinase-independent. Taken together, these results demonstrate that Bmx is a critical downstream target of the constitutively active PI 3-kinase in PTEN-deficient PCa cells and further show that Bmx is recruited by the EGF receptor and ErbB3 and activated in response to their respective ligands. Therefore, Bmx may be a valuable therapeutic target in PCa and other epithelial malignancies in which PI 3-kinase or EGF receptor family pathways are activated.

  9. Tyrosine Kinase Domain Gene Polymorphism of Epidermal Growth Factor Receptor in Gastric Cancer in Northern Iran

    Directory of Open Access Journals (Sweden)

    Jeivad F

    2012-01-01

    Full Text Available Background: Gastric cancer is one of the most common diseases of digestive system with a low 5-year survival rate and metastasis is the main cause of death. Multi-factors, such as changes in molecular pathways and deregulation of cells are involved in the disease development. Epidermal growth factor receptor pathway (EGFR which is associated with cell proliferation and survival can influence cancer development. EGFR function is governed by its genetic polymorphism; thus, we aimed to study the tyrosine kinase domain gene mutations of the receptor in patients with gastric cancer.Methods : In this experimental study, 123 subjects (83 patients with gastric cancer and 40 normal subjects were investigated in north of Iran for EGFR gene polymorphisms during 1 year. Genomic DNA was extracted by DNA extraction kit according to the manufacture's protocol. Polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP and silver staining were performed for investigating EGFR gene polymorphisms. Results : The participants included 72 men and 44 women. Gene polymorphism in exon 18 was present in 10% of the study population but SSCP pattern in exon 19 did not show different migrate bands neither in patients nor in normal subjects.Conclusion: It seems that screening for tyrosine kinas gene polymorphism of epidermal growth factor receptor in patients with gastric cancer and use of tyrosine kinas inhibitors could be useful in the prevention of disease progress and improvement of treatment process for a better quality of life in these patients.

  10. Negative regulation of receptor tyrosine kinases: unexpected links to c-Cbl and receptor ubiquitylation

    Institute of Scientific and Technical Information of China (English)

    Chanan RUBIN; Gal GUR; Yosef YARDEN

    2005-01-01

    Intracellular signals mediated by the family of receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis. Precise control of signal amplitude and duration is critical for the fidelity and robustness of these processes. Activation of receptor tyrosine kinases by their cognate growth factors not only leads to propagation of the signal through various biochemical cascades, but also sets in motion multiple attenuation mechanisms that ultimately terminate the active state. Early attenuators pre-exist prior to receptor activation and they act to limit signal propagation. Subsequently, late attenuators, such as Lrig and Sprouty, are transcriptionally induced and further act to dampen the signal. Central to the process of signaling attenuation is the role of the E3 ubiquitin ligase c-Cbl. While Cblmediated processes of receptor ubiquitylation and endocytosis are relatively well understood, the links of Cbl to other negative regulators are just now beginning to be appreciated. Here we review some emerging interfaces between Cbl and the transcriptionally induced negative regulators Lrig and Sprouty.

  11. Small-Molecule Inhibitors of the Receptor Tyrosine Kinases: Promising Tools for Targeted Cancer Therapies

    Directory of Open Access Journals (Sweden)

    Mohammad Hojjat-Farsangi

    2014-08-01

    Full Text Available Chemotherapeutic and cytotoxic drugs are widely used in the treatment of cancer. In spite of the improvements in the life quality of patients, their effectiveness is compromised by several disadvantages. This represents a demand for developing new effective strategies with focusing on tumor cells and minimum side effects. Targeted cancer therapies and personalized medicine have been defined as a new type of emerging treatments. Small molecule inhibitors (SMIs are among the most effective drugs for targeted cancer therapy. The growing number of approved SMIs of receptor tyrosine kinases (RTKs i.e., tyrosine kinase inhibitors (TKIs in the clinical oncology imply the increasing attention and application of these therapeutic tools. Most of the current approved RTK–TKIs in preclinical and clinical settings are multi-targeted inhibitors with several side effects. Only a few specific/selective RTK–TKIs have been developed for the treatment of cancer patients. Specific/selective RTK–TKIs have shown less deleterious effects compared to multi-targeted inhibitors. This review intends to highlight the importance of specific/selective TKIs for future development with less side effects and more manageable agents. This article provides an overview of: (1 the characteristics and function of RTKs and TKIs; (2 the recent advances in the improvement of specific/selective RTK–TKIs in preclinical or clinical settings; and (3 emerging RTKs for targeted cancer therapies by TKIs.

  12. Effects of 4 multitargeted receptor tyrosine kinase inhibitors on regional hemodynamics in conscious, freely moving rats

    Science.gov (United States)

    Carter, Joanne J.; Fretwell, Laurice V.; Woolard, Jeanette

    2017-01-01

    VEGF inhibitors, including receptor tyrosine kinase inhibitors, are used as adjunct therapies in a number of cancer treatments. An emerging issue with these drugs is that most cause hypertension. To gain insight into the physiological mechanisms involved, we evaluated their regional hemodynamic effects in conscious rats. Male Sprague Dawley rats (350–450 g) were chronically implanted with pulsed Doppler flow probes (renal and mesenteric arteries, and the descending abdominal aorta) and catheters (jugular vein, peritoneal cavity, and distal abdominal aorta). Regional hemodynamics were measured over 4 d, before and after daily administration of cediranib (3 and 6 mg/kg, 3 and 6 mg/kg/h for 1 h, i.v.), sorafenib (10 and 20 mg/kg, 10 and 20 mg kg/h for 1 h, i.v.), pazopanib (30 and100 mg/kg, i.p.), or vandetanib (12.5 and 25 mg/kg, i.p.). All drugs evoked significant increases (P phentolamine and propranolol (each 1 mg/kg/h), suggesting a need for new strategies to overcome them.—Carter, J. J., Fretwell, L. V., Woolard, J. Effects of 4 multitargeted receptor tyrosine kinase inhibitors on regional hemodynamics in conscious, freely moving rats. PMID:27986807

  13. Predominant expression of murine Bmx tyrosine kinase in the granulo-monocytic lineage.

    Science.gov (United States)

    Weil, D; Power, M A; Smith, S I; Li, C L

    1997-12-01

    In the course of systematic cloning of protein tyrosine kinases (PTKs) expressed in hematopoietic stem and progenitor cells, we have identified the murine homologue of human Bmx. It encodes a protein containing the five domains characteristic of the Tec family of cytoplasmic src-related PTKs: pleckstrin homology (PH), Tec homology (TH), src homology 3 and 2 (SH3 and SH2), and tyrosine kinase (TK). In adults, Bmx expression was found primarily in bone marrow and at a lower level in lung and heart. During fetal development it was also found in the spleen at late stage of gestation and in neonates. Analysis of bone marrow subpopulations showed that Bmx was expressed in the progenitor cell population and maturing hematopoietic cells of the granulo/monocytic lineage where expression increased with maturation and differentiation. At the periphery, a high level of Bmx expression was also found in neutrophils and monocytes/macrophages. Bmx expression was not detected in the primitive hematopoietic stem cell population, and cells of the B-, T-, and erythroid-lineages. It was also not detected in most of the cell lines examined. Our results indicate that Bmx is another member of the Btk/Itk/Tec PTK family, which is predominantly expressed in the granulo-monocytic lineage within the hematopoietic system.

  14. Receptor-Tyrosine-Kinase-Targeted Therapies for Head and Neck Cancer

    Directory of Open Access Journals (Sweden)

    Lisa A. Elferink

    2011-01-01

    Full Text Available Molecular therapeutics for treating epidermal growth factor receptor-(EGFR- expressing cancers are a specific method for treating cancers compared to general cell loss with standard cytotoxic therapeutics. However, the finding that resistance to such therapy is common in clinical trials now dampens the initial enthusiasm over this targeted treatment. Yet an improved molecular understanding of other receptor tyrosine kinases known to be active in cancer has revealed a rich network of cross-talk between receptor pathways with a key finding of common downstream signaling pathways. Such cross talk may represent a key mechanism for resistance to EGFR-directed therapy. Here we review the interplay between EGFR and Met and the type 1 insulin-like growth factor receptor (IGF-1R tyrosine kinases, as well as their contribution to anti-EGFR therapeutic resistance in the context of squamous cell cancer of the head and neck, a tumor known to be primarily driven by EGFR-related oncogenic signals.

  15. Measurement of the formation of complexes in tyrosine kinase-mediated signal transduction

    Energy Technology Data Exchange (ETDEWEB)

    Ladbury, John E., E-mail: j.ladbury@biochem.ucl.ac.uk [Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT (United Kingdom)

    2007-01-01

    The use of isothermal titration calorimetry (ITC) provides a full thermodynamic characterization of an interaction in one experiment. The determination of the affinity is an important value; however, the additional layer of information provided by the change in enthalpy and entropy can help in understanding the biology. This is demonstrated with respect to tyrosine kinase-mediated signal transduction. Isothermal titration calorimetry (ITC) provides highly complementary data to high-resolution structural detail. An overview of the methodology of the technique is provided. Ultimately, the correlation of the thermodynamic parameters determined by ITC with structural perturbation observed on going from the free to the bound state should be possible at an atomic level. Currently, thermodynamic data provide some insight as to potential changes occurring on complex formation. Here, this is demonstrated in the context of in vitro quantification of intracellular tyrosine kinase-mediated signal transduction and the issue of specificity of the important interactions. The apparent lack of specificity in the interactions of domains of proteins involved in early signalling from membrane-bound receptors is demonstrated using data from ITC.

  16. Baicalin and Baicalein Inhibit Src Tyrosine Kinase and Production of IL-6

    Directory of Open Access Journals (Sweden)

    Dubravko Jelić

    2016-01-01

    Full Text Available Flavonoids play an important role in the treatment of various diseases, as they are able to inhibit reactive oxygen species, which cause damage to cells and tissues which may lead to increased risk of inflammatory diseases. Baicalin and baicalein, two flavonoids found in the roots of Scutellaria baicalensis, in the leaves of Thymus vulgaris and Oroxylum indicum, were tested for their anti-inflammatory activity as well as for their cytotoxicity. Thereby the two compounds were investigated on Src tyrosine kinase inhibition and inhibition of production of interleukin (IL-6 in lipopolysaccharide- (LPS- stimulated THP-1 cells. Additionally, the THP-1 cell line was used for the determination of the cytotoxicity. Both baicalin and baicalein showed some anti-inflammatory properties, while baicalein turned out to be the more active compound with higher inhibitory activities on both Src tyrosine kinase and production of cytokine IL-6. Baicalin and baicalein showed no signs of cytotoxicity in the MTS cytotoxicity assay in THP-1 cells.

  17. Haemophilus ducreyi targets Src family protein tyrosine kinases to inhibit phagocytic signaling.

    Science.gov (United States)

    Mock, Jason R; Vakevainen, Merja; Deng, Kaiping; Latimer, Jo L; Young, Jennifer A; van Oers, Nicolai S C; Greenberg, Steven; Hansen, Eric J

    2005-12-01

    Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi. Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi, but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fcgamma receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi. This is the first example of a bacterial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.

  18. Dynamics of the Tec-family tyrosine kinase SH3 domains.

    Science.gov (United States)

    Roberts, Justin M; Tarafdar, Sreya; Joseph, Raji E; Andreotti, Amy H; Smithgall, Thomas E; Engen, John R; Wales, Thomas E

    2016-04-01

    The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X-ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src-family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src-family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher-order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra- and intermolecular binding assays of proteins containing the domains.

  19. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    Directory of Open Access Journals (Sweden)

    Ayrapetov Marina K

    2005-11-01

    Full Text Available Abstract Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1 binds to ATP, and the other (M2 acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator.

  20. 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.

  1. Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn

    OpenAIRE

    1992-01-01

    Recent observations suggest that the src-related tyrosine protein kinase p59fyn may be involved in antigen-induced T lymphocyte activation. As a result of alternative splicing, p59fyn exists as two isoforms that differ exclusively within a short sequence spanning the end of the Src Homology 2 (SH2) region and the beginning of the tyrosine protein kinase domain. While one p59fyn isoform (fynB) is highly expressed in brain, the alternative product (fynT) is principally found in T lymphocytes. T...

  2. p59fyn tyrosine kinase associates with multiple T-cell receptor subunits through its unique amino-terminal domain.

    OpenAIRE

    1992-01-01

    Several lines of evidence link the protein tyrosine kinase p59fyn to the T-cell receptor. The molecular basis of this interaction has not been established. Here we show that the tyrosine kinase p59fyn can associate with chimeric proteins that contain the cytoplasmic domains of CD3 epsilon, gamma, zeta (zeta), and eta. Mutational analysis of the zeta cytoplasmic domain demonstrated that the membrane-proximal 41 residues of zeta are sufficient for p59fyn binding and that at least two p59fyn bin...

  3. The c-Met receptor tyrosine kinase inhibitor MP470 radiosensitizes glioblastoma cells

    Directory of Open Access Journals (Sweden)

    Bearss David

    2009-12-01

    Full Text Available Abstract Purpose Glioblastoma multiforme (GBM is resistant to current cytotoxic therapies, in part because of enhanced DNA repair. Activation of the receptor tyrosine kinase c-Met has been shown to protect cancer cells from DNA damage. We hypothesized that inhibiting c-Met would decrease this protection and thus sensitize resistant tumor cells to the effects of radiation therapy. Materials and methods Eight human GBM cell lines were screened for radiosensitivity to the small-molecule c-Met inhibitor MP470 with colony-count assays. Double-strand (ds DNA breaks was quantified by using antibodies to gamma H2AX. Western blotting demonstrate expression of RAD51, glycogen synthase kinase (GSK-3β, and other proteins. A murine xenograft tumor flank model was used for in vivo radiosensitization studies. Results MP470 reduced c-Met phosphorylation and enhanced radiation-induced cell kill by 0.4 logs in SF767 cells. Cells pretreated with MP470 had more ds DNA damage than cells treated with radiation alone. Mechanistically, MP470 was shown to inhibit dsDNA break repair and increase apoptosis. MP470 influences various survival and DNA repair related proteins such as pAKT, RAD51 and GSK3β. In vivo, the addition of MP470 to radiation resulted in a tumor-growth-delay enhancement ratio of 2.9 over radiation alone and extended survival time. Conclusions GBM is a disease site where radiation is often used to address both macroscopic and microscopic disease. Despite attempts at dose escalation outcomes remain poor. MP470, a potent small-molecule tyrosine kinase inhibitor of c-Met, radiosensitized several GBM cell lines both in vitro and in vivo, and may help to improve outcomes for patients with GBM.

  4. Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation

    Science.gov (United States)

    Vasu, Dudipeta; Kumar, Pasupuleti Santhosh; Prasad, Uppu Venkateswara; Swarupa, Vimjam; Yeswanth, Sthanikam; Srikanth, Lokanathan; Sunitha, Manne Mudhu; Choudhary, Abhijith; Krishna Sarma, Potukuchi Venkata Gurunadha

    2017-01-01

    Background: When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. Methods: Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK, and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. Results: Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. Conclusion: The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and the progress of infection. PMID:27695030

  5. The Activation of c-Src Tyrosine Kinase: Conformational Transition Pathway and Free Energy Landscape.

    Science.gov (United States)

    Fajer, Mikolai; Meng, Yilin; Roux, Benoît

    2016-10-28

    Tyrosine kinases are important cellular signaling allosteric enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Their activity must be tightly controlled, and malfunction can lead to a variety of diseases, particularly cancer. The nonreceptor tyrosine kinase c-Src, a prototypical model system and a representative member of the Src-family, functions as complex multidomain allosteric molecular switches comprising SH2 and SH3 domains modulating the activity of the catalytic domain. The broad picture of self-inhibition of c-Src via the SH2 and SH3 regulatory domains is well characterized from a structural point of view, but a detailed molecular mechanism understanding is nonetheless still lacking. Here, we use advanced computational methods based on all-atom molecular dynamics simulations with explicit solvent to advance our understanding of kinase activation. To elucidate the mechanism of regulation and self-inhibition, we have computed the pathway and the free energy landscapes for the "inactive-to-active" conformational transition of c-Src for different configurations of the SH2 and SH3 domains. Using the isolated c-Src catalytic domain as a baseline for comparison, it is observed that the SH2 and SH3 domains, depending upon their bound orientation, promote either the inactive or active state of the catalytic domain. The regulatory structural information from the SH2-SH3 tandem is allosterically transmitted via the N-terminal linker of the catalytic domain. Analysis of the conformational transition pathways also illustrates the importance of the conserved tryptophan 260 in activating c-Src, and reveals a series of concerted events during the activation process.

  6. Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption.

    Science.gov (United States)

    McGinnis, Lynda K; Luo, Jinping; Kinsey, William H

    2013-04-01

    Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

  7. Small Molecule Tyrosine Kinase Inhibitors of ErbB2/HER2/Neu in the Treatment of Aggressive Breast Cancer

    Directory of Open Access Journals (Sweden)

    Richard L. Schroeder

    2014-09-01

    Full Text Available The human epidermal growth factor receptor 2 (HER2 is a member of the erbB class of tyrosine kinase receptors. These proteins are normally expressed at the surface of healthy cells and play critical roles in the signal transduction cascade in a myriad of biochemical pathways responsible for cell growth and differentiation. However, it is widely known that amplification and subsequent overexpression of the HER2 encoding oncogene results in unregulated cell proliferation in an aggressive form of breast cancer known as HER2-positive breast cancer. Existing therapies such as trastuzumab (Herceptin® and lapatinib (Tyverb/Tykerb®, a monoclonal antibody inhibitor and a dual EGFR/HER2 kinase inhibitor, respectively, are currently used in the treatment of HER2-positive cancers, although issues with high recurrence and acquired resistance still remain. Small molecule tyrosine kinase inhibitors provide attractive therapeutic targets, as they are able to block cell signaling associated with many of the proposed mechanisms for HER2 resistance. In this regard we aim to present a review on the available HER2 tyrosine kinase inhibitors, as well as those currently in development. The use of tyrosine kinase inhibitors as sequential or combinatorial therapeutic strategies with other HER family inhibitors is also discussed.

  8. Phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) serine-511 by the combined action of tyrosine kinases and CK2: the implication of tyrosine-512 and phenylalanine-508.

    Science.gov (United States)

    Cesaro, Luca; Marin, Oriano; Venerando, Andrea; Donella-Deana, Arianna; Pinna, Lorenzo A

    2013-12-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) harbors, close to Phe-508, whose deletion is the commonest cause of cystic fibrosis, a conserved potential CK2 phospho-acceptor site (Ser511), which however is not susceptible to phosphorylation by CK2. To shed light on this apparent paradox, a series of systematically substituted peptides encompassing Ser511 were assayed for their ability to be phosphorylated. The main outcomes of our study are the following: (a) Tyr512 plays a prominent role as a negative determinant as its replacement by Ala restores Ser511 phosphorylation by CK2; (b) an even more pronounced phosphorylation of Ser511 is promoted if Tyr512 is replaced by phospho-tyrosine instead of alanine; (c) Tyr512 and, to a lesser extent, Tyr515 are readily phosphorylated by Lyn, a protein tyrosine kinase of the Src family, in a manner which is enhanced by the concomitant Phe508 deletion. Collectively taken, our data, in conjunction with the notion that Tyr515 is phosphorylated in vivo, disclose the possibility that CFTR Ser511 can be phosphorylated by the combined action of tyrosine kinases and CK2 and disclose a new mechanism of hierarchical phosphorylation where the role of the priming kinase is that of removing negative determinant(s).

  9. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    Energy Technology Data Exchange (ETDEWEB)

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F. (Sunesis); (Biogen)

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  10. Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling.

    Science.gov (United States)

    Friedman, Adam A; Tucker, George; Singh, Rohit; Yan, Dong; Vinayagam, Arunachalam; Hu, Yanhui; Binari, Richard; Hong, Pengyu; Sun, Xiaoyun; Porto, Maura; Pacifico, Svetlana; Murali, Thilakam; Finley, Russell L; Asara, John M; Berger, Bonnie; Perrimon, Norbert

    2011-10-25

    Characterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification-mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type- and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine-protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.

  11. Bruton's tyrosine kinase regulates the activation of gene rearrangements at the lambda light chain locus in precursor B cells in the mouse

    NARCIS (Netherlands)

    G.M. Dingjan (Gemma); S. Middendorp; K. Dahlenborg; A. Maas (Alex); R.W. Hendriks (Rudi); F.G. Grosveld (Frank)

    2001-01-01

    textabstractBruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase involved in precursor B (pre-B) cell receptor signaling. Here we demonstrate that Btk-deficient mice have an approximately 50% reduction in the frequency of immunoglobulin (Ig) lambda light chai

  12. Identification and regulation of receptor tyrosine kinases Rse and Mer and their ligand Gas6 in testicular somatic cells.

    Science.gov (United States)

    Chan, M C; Mather, J P; McCray, G; Lee, W M

    2000-01-01

    Receptor tyrosine kinases act to convey extracellular signals to intracellular signaling pathways and ultimately control cell proliferation and differentiation. Rse, Axl, and Mer belong to a newly identified family of cell adhesion molecule-related receptor tyrosine kinase. They bind the vitamin K-dependent protein growth arrest-specific gene 6 (Gas6), which is also structurally related to the anticoagulation factor Protein S. The aim of this study is to investigate the possible role of Rse/Axl/Mer tyrosine kinase receptors and their ligand in regulating testicular functions. Gene expression of Rse, Axl, Mer, and Gas6 in the testis was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis. The results indicated that receptors Rse and Mer and the ligand Gas6 were expressed in the rat endothelial cell line (TR1), mouse Leydig cell line (TM3), rat peritubular myoid cell line (TRM), mouse Sertoli cell line (TM4), and primary rat Sertoli cells. Axl was not expressed in the testicular somatic cells by RT-PCR or Northern blot analysis. The highest level of expression of Gas6 messenger RNA (mRNA) was observed in the Sertoli cells, and its expression was responsive to the addition of forskolin in vitro. The effects of serum, insulin, and transferrin on Gas6 expression by TM4 cells were examined. It was shown that they all exhibited an up-regulating effect on Gas6 expression. The forskolin-stimulated Gas6 expression was accompanied by an increase in tyrosine phosphorylation of the Rse receptor in vitro, suggesting that Gas6 may exhibit an autocrine effect in the Sertoli cells through multiple tyrosine kinase receptors. Our studies so far have demonstrated that tyrosine kinase receptors Rse and Mer and their ligand Gas6 are widely expressed in the testicular somatic cell lines and may play a marked role in promoting testicular cell survival.

  13. Comparative VEGF receptor tyrosine kinase modeling for the development of highly specific inhibitors of tumor angiogenesis.

    Science.gov (United States)

    Schmidt, Ulrike; Ahmed, Jessica; Michalsky, Elke; Hoepfner, Michael; Preissner, Robert

    2008-01-01

    The Vascular Endothelial Growth Factor receptors (VEGF-Rs) play a significant role in tumor development and tumor angiogenesis and are therefore interesting targets in cancer therapy. Targeting the VEGF-R is of special importance as the feed of the tumor has to be reduced. In general, this can be carried out by inhibiting the tyrosine kinase function of the VEGF-R. Nevertheless, there arise some problems with the specificity of known kinase inhibitors: they bind to the ATP-binding site and inhibit a number of kinases, moreover the so far most specific inhibitors act at least on these three major types of VEGF-Rs: Flt-1, Flk-1/KDR, Flt-4. The goal is a selective VEGF-R-2 (Flk-1/KDR) inhibitor, because this receptor triggers rather unspecific signals from VEGF-A, -C, -D and -E. Here, we describe a protocol starting from an established inhibitor (Vatalanib) with 2D-/3D-searching and property filtering of the in silico screening hits and the "negative docking approach". With this approach we were able to identify a compound, which shows a fourfold higher reduction of the proliferation rate of endothelial cells compared to the reduction effect of the lead structure.

  14. Using 2 nd generation tyrosine kinase inhibitors in frontline management of chronic phase chronic myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Vishal Jayakar

    2014-01-01

    Full Text Available Choices in medicine come with responsibility. With several TKI′s (Tyrosine kinase inhibitors available for front-line management of CML (Chronic Myeloid Leukemia, an astute clinician has to personalise, rationalise and take a pragmatic approach towards selection of the best drug for the ′patient in question′. Though it is hotly debated as to which TKI will triumph, the truth of this debate lies in individualising treatment rather than a general ′all size fits all′ approach with imatinib. I personally believe that the second generation TKI′s will suit most patient clinical profiles rather than prescribing imatinib to all and I have strived to make a strong case for them in front line treatment of CML. Though Imatinib may remain the first line choice for some patients, my efforts in this debate are mainly geared towards breaking the myth that imatinib is the sole ′block buster′ on the CML landscape

  15. Bruton's tyrosine kinase: from X-linked agammaglobulinemia toward targeted therapy for B-cell malignancies.

    Science.gov (United States)

    Ponader, Sabine; Burger, Jan A

    2014-06-10

    Discovery of Bruton's tyrosine kinase (BTK) mutations as the cause for X-linked agammaglobulinemia was a milestone in understanding the genetic basis of primary immunodeficiencies. Since then, studies have highlighted the critical role of this enzyme in B-cell development and function, and particularly in B-cell receptor signaling. Because its deletion affects mostly B cells, BTK has become an attractive therapeutic target in autoimmune disorders and B-cell malignancies. Ibrutinib (PCI-32765) is the most advanced BTK inhibitor in clinical testing, with ongoing phase III clinical trials in patients with chronic lymphocytic leukemia and mantle-cell lymphoma. In this article, we discuss key discoveries related to BTK and clinically relevant aspects of BTK inhibitors, and we provide an outlook into clinical development and open questions regarding BTK inhibitor therapy.

  16. Mutations of Bruton's tyrosine kinase gene in Brazilian patients with X-linked agammaglobulinemia.

    Science.gov (United States)

    Ramalho, V D; Oliveira Júnior, E B; Tani, S M; Roxo Júnior, P; Vilela, M M S

    2010-09-01

    Mutations in Bruton's tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA), which is characterized by recurrent bacterial infections, profound hypogammaglobulinemia, and decreased numbers of mature B cells in peripheral blood. We evaluated 5 male Brazilian patients, ranging from 3 to 10 years of age, from unrelated families, whose diagnosis was based on recurrent infections, markedly reduced levels of IgM, IgG and IgA, and circulating B cell numbers <2%. BTK gene analysis was carried out using PCR-SSCP followed by sequencing. We detected three novel (Ala347fsX55, I355T, and Thr324fsX24) and two previously reported mutations (Q196X and E441X). Flow cytometry revealed a reduced expression of BTK protein in patients and a mosaic pattern of BTK expression was obtained from mothers, indicating that they were XLA carriers.

  17. Mutations of Bruton's tyrosine kinase gene in Brazilian patients with X-linked agammaglobulinemia

    Directory of Open Access Journals (Sweden)

    V.D. Ramalho

    2010-09-01

    Full Text Available Mutations in Bruton's tyrosine kinase (BTK gene are responsible for X-linked agammaglobulinemia (XLA, which is characterized by recurrent bacterial infections, profound hypogammaglobulinemia, and decreased numbers of mature B cells in peripheral blood. We evaluated 5 male Brazilian patients, ranging from 3 to 10 years of age, from unrelated families, whose diagnosis was based on recurrent infections, markedly reduced levels of IgM, IgG and IgA, and circulating B cell numbers <2%. BTK gene analysis was carried out using PCR-SSCP followed by sequencing. We detected three novel (Ala347fsX55, I355T, and Thr324fsX24 and two previously reported mutations (Q196X and E441X. Flow cytometry revealed a reduced expression of BTK protein in patients and a mosaic pattern of BTK expression was obtained from mothers, indicating that they were XLA carriers.

  18. Bmx tyrosine kinase transgene induces skin hyperplasia, inflammatory angiogenesis, and accelerated wound healing.

    Science.gov (United States)

    Paavonen, Karri; Ekman, Niklas; Wirzenius, Maria; Rajantie, Iiro; Poutanen, Matti; Alitalo, Kari

    2004-09-01

    The Bmx gene, a member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in arterial endothelium and in certain hematopoietic and epithelial cells. Previous in vitro studies have implicated Bmx signaling in cell migration and survival and suggested that it contributes to the progression of prostate carcinomas. However, the function of Bmx in normal tissues in vivo is unknown. We show here that Bmx expression is induced in skin keratinocytes during wound healing. To analyze the role of Bmx in epidermal keratinocytes in vivo, we generated transgenic mice overexpressing Bmx in the skin. We show that Bmx overexpression accelerates keratinocyte proliferation and wound reepithelialization. Bmx expression also induces chronic inflammation and angiogenesis in the skin, and gene expression profiling suggests that this occurs via cytokine-mediated recruitment of inflammatory cells. Our studies provide the first data on Bmx function in vivo and form the basis of evaluation of its role in epithelial neoplasia.

  19. The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity

    Science.gov (United States)

    Chan, Pamela Y.; Carrera Silva, Eugenio A.; De Kouchkovsky, Dimitri; Joannas, Leonel D.; Hao, Liming; Hu, Donglei; Huntsman, Scott; Eng, Celeste; Licona-Limón, Paula; Weinstein, Jason S.; Herbert, De’Broski R.; Craft, Joseph E.; Flavell, Richard A.; Repetto, Silvia; Correale, Jorge; Burchard, Esteban G.; Torgerson, Dara G.; Ghosh, Sourav; Rothlin, Carla V.

    2016-01-01

    Host responses against metazoan parasites or an array of environmental substances elicit type 2 immunity. Despite its protective function, type 2 immunity also drives allergic diseases. The mechanisms that regulate the magnitude of the type 2 response remain largely unknown. Here, we show that genetic ablation of a receptor tyrosine kinase encoded by Tyro3 in mice or the functional neutralization of its ortholog in human dendritic cells resulted in enhanced type 2 immunity. Furthermore, the TYRO3 agonist PROS1 was induced in T cells by the quintessential type 2 cytokine, interleukin-4. T cell–specific Pros1 knockouts phenocopied the loss of Tyro3. Thus, a PROS1-mediated feedback from adaptive immunity engages a rheostat, TYRO3, on innate immune cells to limit the intensity of type 2 responses. PMID:27034374

  20. Tyrosine kinase inhibitors in the treatment of systemic sclerosis: from animal models to clinical trials.

    Science.gov (United States)

    Iwamoto, Naoki; Distler, Jörg H W; Distler, Oliver

    2011-02-01

    Efficient antifibrotic therapies are not available for patients with systemic sclerosis (SSc). This review summarizes the current preclinical and clinical evidence for imatinib and related tyrosine kinase inhibitors as potential antifibrotic therapies for SSc and other fibrotic diseases. In experimental models of SSc, imatinib, nilotinib, and dasatinib demonstrated strong antifibrotic effects. Imatinib not only prevented fibrosis in the bleomycin-induced model of dermal fibrosis and the tight skin mouse-1 model but also reduced established fibrosis in a modified bleomycin model. Open-label, proof-of-concept trials in SSc showed moderate effects on skin fibrosis, biological measures of skin fibrosis, and lung fibrosis compared with baseline measures. However, whether this reflects the natural course of the disease or is a result of treatment effects is unclear and needs to be analyzed in larger, multicenter, randomized, placebo-controlled trials. Toxicity is expected from cancer trials with frequent mild to moderate adverse events.

  1. An overview of the binding models of FGFR tyrosine kinases in complex with small molecule inhibitors.

    Science.gov (United States)

    Cheng, Weiyan; Wang, Mixiang; Tian, Xin; Zhang, Xiaojian

    2017-01-27

    The fibroblast growth factor receptor (FGFR) family receptor tyrosine kinase (RTK) includes four structurally related members, termed as FGFR1, FGFR2, FGFR3, and FGFR4. Given its intimate role in the progression of several solid tumors, excessive FGFR signaling provides an opportunity for anticancer therapy. Along with extensive pharmacological studies validating the therapeutic potential of targeting the FGFRs for cancer treatment, co-crystal structures of FGFRs/inhibitors are continuously coming up to study the mechanism of actions and explore new inhibitors. Herein, we review the reported co-crystals of FGFRs in complex with the corresponding inhibitors, main focusing our attention on the binding models and the pharmacological activities of the inhibitors.

  2. Ibrutinib: a novel Bruton's tyrosine kinase inhibitor with outstanding responses in patients with chronic lymphocytic leukemia.

    Science.gov (United States)

    Barrientos, Jacqueline; Rai, Kanti

    2013-08-01

    New treatment options are urgently needed for patients with relapsed chronic lymphocytic leukemia (CLL) who fail to respond to currently available therapies or cannot achieve a sustained response. Moreover, targeted agents with less myelotoxicity are necessary to treat patients with multiple comorbidities who would otherwise be unable to tolerate standard regimens. Ibrutinib, a Bruton's tyrosine kinase inhibitor, has shown highly encouraging results in phase I/II trials in patients with treatment-naive, relapsed and refractory CLL even in the presence of high risk disease or poor prognostic markers. In phase I/II trials, ibrutinib 420 mg or 840 mg - given continuously as single agent or at a dose of 420 mg daily in combination with a monoclonal antibody or chemoimmunotherapy - has been associated with high response rates and durable clinical remissions. Phase II and III trials are currently under way for treatment-naive patients, relapsed/refractory patients, and for those patients harboring a 17p deletion.

  3. Greater Sensitivity of Blood Pressure Than Renal Toxicity to Tyrosine Kinase Receptor Inhibition With Sunitinib

    DEFF Research Database (Denmark)

    Lankhorst, Stephanie; Baelde, Hans J; Kappers, Mariëtte H W

    2015-01-01

    of these side effects. Normotensive Wistar Kyoto rats were exposed to 3 different doses of sunitinib or vehicle. After 8 days, rats were euthanized. Telemetrically measured blood pressure rose dose dependently, from 13 to 30 mm Hg. Proteinuria was present at all doses, but a rise in cystatin C occurred only...... histological abnormalities with the low dose. Podocyte number per glomerular circumference did not change. Glomerular nephrin, Neph1, podocin, and endothelin-converting enzyme gene expression were downregulated in a dose-dependent manner. We conclude that the sunitinib-induced rise in blood pressure requires......Hypertension and renal injury are off-target effects of sunitinib, a tyrosine kinase receptor inhibitor used for the treatment of various tumor types. Importantly, these untoward effects are accompanied by activation of the endothelin system. Here, we set up a study to explore the dose dependency...

  4. Preparation and Characterization of {sup 177}Lu Labeled Antibody against Tyrosine Kinase Receptor Her2

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So-Young; Hong, Young-Don; Choi, Sun-Ju [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2008-05-15

    The tyrosine kinase receptor Her2, also known in humans as erbB2, is a member of the epidermal growth factor receptor (EGFR or erbB1) family. The Her2 is highly expressed in many cancer types and over expressed in approximately 30% of all primary breast cancer. Overexpression of Her2 is associated with a poor prognosis. Her2 is a suitable target because it involves an extracellular domain that can be targeted by antibodies produced by B cells. Based on these advantages, we tried to prepare the {sup 177}Lu labeled Her2 antibody. This radioimmunoconjugate could act by not only blocking the Her2 signalling pathway using antibody but also killing the tumour cell using {beta} energy of {sup 177}Lu.

  5. Non-radioisotopic method for the in vitro measurement of EGF receptor tyrosine kinase

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A non-radioisotopic method was developed for the assay ofepidermal growth factor receptor (EGFR). A peptide with twenty amino acid residues around Tyr 1173, the major phosphorylation site of EGFR, was cloned as a GST fusion protein and used as substrate. Anti-phosphotyrosine monoclonal antibody PY99 was used for the determination of the extent of phosphorylation. Both the specificity and the sensitivity were substantially higher than that of the existing method. Km value of the fusion protein is much lower (10 (mol/L) than that of the synthetic peptide (110 (mol/L). The method can be applied to the measurement of the tyrosine kinase activity of c-erb B2 (Neu/HER2).

  6. The C. elegans ROR receptor tyrosine kinase, CAM-1, non-autonomously inhibits the Wnt pathway.

    Science.gov (United States)

    Green, Jennifer L; Inoue, Takao; Sternberg, Paul W

    2007-11-01

    Inhibitors of Wnt signaling promote normal development and prevent cancer by restraining when and where the Wnt pathway is activated. ROR proteins, a class of Wnt-binding receptor tyrosine kinases, inhibit Wnt signaling by an unknown mechanism. To clarify how RORs inhibit the Wnt pathway, we examined the relationship between Wnts and the sole C. elegans ROR homolog, cam-1, during C. elegans vulval development, a Wnt-regulated process. We found that loss and overexpression of cam-1 causes reciprocal defects in Wnt-mediated cell-fate specification. Our molecular and genetic analyses revealed that the CAM-1 extracellular domain (ECD) is sufficient to non-autonomously antagonize multiple Wnts, suggesting that the CAM-1/ROR ECD sequesters Wnts. A sequestration model is supported by our findings that the CAM-1 ECD binds to several Wnts in vitro. These results demonstrate how ROR proteins help to refine the spatial pattern of Wnt activity in a complex multicellular environment.

  7. Pyrrolo[2,3-b]pyridine derivatives as potent Bruton's tyrosine kinase inhibitors.

    Science.gov (United States)

    Zhao, Xinge; Huang, Wei; Wang, Yazhou; Xin, Minhang; Jin, Qiu; Cai, Jianfeng; Tang, Feng; Zhao, Yong; Xiang, Hua

    2015-08-01

    A series of pyrrolo[2,3-b]pyridine-based derivatives were designed as potent Bruton's tyrosine kinase (BTK) inhibitors by using a scaffold-hopping strategy. Structure-activity relationship studies identified five compounds (3n, 3p, 3q, 3r, and 3s) with IC50 of less than 10nM in BTK enzyme assay and five compounds (3m, 3n, 3o, 3p, and 3t) with IC50 of less than 20 nM in Ramos cell assay. As one of the most potent inhibitors, compound 3p exhibited superior activity to that of compound 1 (RN486) and pyrrolo[2,3-d]pyrimidine derivative 2 in both BTK enzymatic (IC50=6.0 nM) and cellular inhibition (IC50=14 nM) assays. In addition, 3p displayed favorable overall pharmacokinetic profiles compared with 1 and 2.

  8. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2012-10-01

    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  9. Activation of tyrosine kinase c-Abl contributes to α-synuclein–induced neurodegeneration

    Science.gov (United States)

    Lee, Su Hyun; Kim, Donghoon; Karuppagounder, Senthilkumar S.; Kumar, Manoj; Mao, Xiaobo; Shin, Joo Ho; Lee, Yunjong; Pletnikova, Olga; Troncoso, Juan C.; Dawson, Valina L.; Dawson, Ted M.; Ko, Han Seok

    2016-01-01

    Aggregation of α-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and α-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote α-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates α-synuclein–induced neuropathology. In mice expressing a human α-synucleinopathy–associated mutation (hA53Tα-syn mice), deletion of the gene encoding c-Abl reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53Tα-syn mice accelerated α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 α-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 α-synuclein in brain tissues and Lewy bodies of PD patients compared with age-matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of α-synuclein at tyrosine 39 enhances α-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in α-synuclein–induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 α-synuclein is a potential disease indicator for PD and related α-synucleinopathies. PMID:27348587

  10. The receptor tyrosine kinase FGFR4 negatively regulates NF-kappaB signaling.

    Directory of Open Access Journals (Sweden)

    Kristine A Drafahl

    Full Text Available BACKGROUND: NFκB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs, including the Fibroblast Growth Factor Receptors (FGFRs are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFκB activation has not been previously described, although FGFs have been known to antagonize TNFα-induced apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate an interaction between FGFR4 and IKKβ (Inhibitor of NFκB Kinase β subunit, an essential component in the NFκB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen [1] and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKβ in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFα (Tumor Necrosis Factor α to activate NFκB pathways, FGFR4 activation results in significant inhibition of NFκB signaling as measured by decreased nuclear NFκB localization, by reduced NFκB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKβ kinase activity towards the substrate GST-IκBα in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFα-treated DU145 prostate cancer cells also leads to a decrease in IKKβ activity, concomitant reduction in NFκB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFα treatment of DU145 cells, in comparison with TNFα alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFκB signaling. CONCLUSIONS/SIGNIFICANCE: These results identify a compelling link between FGFR4 signaling and the NFκB pathway, and reveal that FGFR4 activation leads to a negative effect on NFκB signaling

  11. Mutation pattern in the Bruton's tyrosine kinase gene in 26 unrelated patients with X-linked agammaglobulinemia

    DEFF Research Database (Denmark)

    Vorechovský, I; Luo, L; Hertz, Jens Michael

    1997-01-01

    Mutation pattern was characterized in the Bruton's tyrosine kinase gene (BTK) in 26 patients with X-linked agammaglobulinemia, the first described immunoglobulin deficiency, and was related to BTK expression. A total of 24 different mutations were identified. Most BTK mutations were found to resu...

  12. Bmx tyrosine kinase has a redundant function downstream of angiopoietin and vascular endothelial growth factor receptors in arterial endothelium.

    Science.gov (United States)

    Rajantie, I; Ekman, N; Iljin, K; Arighi, E; Gunji, Y; Kaukonen, J; Palotie, A; Dewerchin, M; Carmeliet, P; Alitalo, K

    2001-07-01

    The Bmx gene, a member of the Tec tyrosine kinase gene family, is known to be expressed in subsets of hematopoietic and endothelial cells. In this study, mice were generated in which the first coding exon of the Bmx gene was replaced with the lacZ reporter gene by a knock-in strategy. The homozygous mice lacking Bmx activity were fertile and had a normal life span without an obvious phenotype. Staining of their tissues using beta-galactosidase substrate to assess the sites of Bmx expression revealed strong signals in the endothelial cells of large arteries and in the endocardium starting between days 10.5 and 12.5 of embryogenesis and continuing in adult mice, while the venular endothelium showed a weak signal only in the superior and inferior venae cavae. Of the five known endothelial receptor tyrosine kinases tested, activated Tie-2 induced tyrosyl phosphorylation of the Bmx protein and both Tie-2 and vascular endothelial growth factor receptor 1 (VEGFR-1) stimulated Bmx tyrosine kinase activity. Thus, the Bmx tyrosine kinase has a redundant role in arterial endothelial signal transduction downstream of the Tie-2 and VEGFR-1 growth factor receptors.

  13. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    Science.gov (United States)

    Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

  14. Roles of mitochondrial Src tyrosine kinase and zinc in nitric oxide-induced cardioprotection against ischemia/reperfusion injury.

    Science.gov (United States)

    Zhang, Y; Xing, F; Zheng, H; Xi, J; Cui, X; Xu, Z

    2013-07-01

    While nitric oxide (NO) induces cardioprotection by targeting the mitochondrial permeability transition pore (mPTP), the precise mitochondrial signaling events that mediate the action of NO remain unclear. The purpose of this study was to test whether NO induces cardioprotection against ischemia/reperfusion by inhibiting oxidative stress through mitochondrial zinc and Src tyrosine kinase. The NO donor S-nitroso-N-acetyl penicillamine (SNAP) given before the onset of ischemia reduced cell death in rat cardiomyocytes subjected to simulated ischemia/reperfusion, and this was abolished by the zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and the Src tyrosine kinase inhibitor PP2. SNAP also prevented loss of mitochondrial membrane potential (ΔΨm) at reperfusion, an effect that was blocked by TPEN and PP2. SNAP increased mitochondrion-free zinc upon reperfusion and enhanced mitochondrial Src phosphorylation in a zinc-dependent manner. SNAP inhibited both mitochondrial complex I activity and mitochondrial reactive oxygen species (ROS) generation at reperfusion through zinc and Src tyrosine kinase. Finally, the anti-infarct effect of SNAP was abrogated by TPEN and PP2 applied at reperfusion in isolated rat hearts. In conclusion, NO induces cardioprotection at reperfusion by targeting mitochondria through attenuation of oxidative stress resulted from the inhibition of complex I at reperfusion. Activation of mitochondrial Src tyrosine kinase by zinc may account for the inhibition of complex I.

  15. Myeloproliferative Neoplasms Associated with Mutation in JAK2V617F and Tyrosine Kinase Inhibitors as Therapeutic Strategy

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    Kaveh Tari

    2015-04-01

    Full Text Available MPNs including a heterogeneous group of clonal or oligoclonal hamtopathies characterized by proliferation and accumulation of mature myeloid cells. JAK2 tyrosine kinase mutation is the most common molecular lesion identified in 90% of cases. JAK2 is involved in EPO signaling pathway, and mutations in it lead to EPO-independent spontaneous phosphorylation. Most tyrosine kinase inhibitors (TKI are small molecules that compete with ATP for binding the ATP-binding site in tyrosine kinase domains, since ATP is a source of phosphate groups used by Tks to phosphorylate the target protein.there are many TKI agent that are studing for treatment of the MPNs with JAK2 tyrosine kinase mutation.the most important TKI drugs including CEP701, CYT387, LY2784544, SB1518, TG101348, XL019, INCB18424. Most important mechanism of them are reduse the splenomegaly, improvement of constitutional symptoms(improvement of bone marrow fibrosis and anemia. Although this drugs are useful but they have some side effect that common of them including Gastrointestinal disease (GI, diarrhea, nausea and vomiting, anemia, thrombocytopenia, thrombosis, leukocytosis, thrombocytosis, peripheral neuropathy, transient loss of blood pressure and lightheadedness.

  16. Ligand-based receptor tyrosine kinase partial agonists: New paradigm for cancer drug discovery?

    Science.gov (United States)

    Riese, David J.

    2010-01-01

    Introduction Receptor tyrosine kinases (RTKs) are validated targets for oncology drug discovery and several RTK antagonists have been approved for the treatment of human malignancies. Nonetheless, the discovery and development of RTK antagonists has lagged behind the discovery and development of agents that target G-protein coupled receptors. In part, this is because it has been difficult to discover analogs of naturally-occurring RTK agonists that function as antagonists. Areas covered Here we describe ligands of ErbB receptors that function as partial agonists for these receptors, thereby enabling these ligands to antagonize the activity of full agonists for these receptors. We provide insights into the mechanisms by which these ligands function as antagonists. We discuss how information concerning these mechanisms can be translated into screens for novel small molecule- and antibody-based antagonists of ErbB receptors and how such antagonists hold great potential as targeted cancer chemotherapeutics. Expert opinion While there have been a number of important key findings into this field, the identification of the structural basis of ligand functional specificity is still of the greatest importance. While it is true that, with some notable exceptions, peptide hormones and growth factors have not proven to be good platforms for oncology drug discovery; addressing the fundamental issues of antagonistic partial agonists for receptor tyrosine kinases has the potential to steer oncology drug discovery in new directions. Mechanism based approaches are now emerging to enable the discovery of RTK partial agonists that may antagonize both agonist-dependent and –independent RTK signaling and may hold tremendous promise as targeted cancer chemotherapeutics. PMID:21532939

  17. Hepatitis B Virus Reactivation during Treatment with Multi-Tyrosine Kinase Inhibitor for Hepatocellular Carcinoma

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    Satoshi Shiba

    2012-09-01

    Full Text Available Hepatitis B virus (HBV reactivation is well documented in individuals with cancer who receive certain cytotoxic or immunosuppressive therapies including rituximab treatment. As a general rule, the risk is greatest upon withdrawal of chemotherapy. The risk ranges from approximately 20 to 50% among HBsAg-positive carriers. A 67-year-old man was diagnosed with inoperable multiple hepatocellular carcinoma accompanied by an increase in alpha-fetoprotein and protein induced by vitamin K absence or antagonist II level. Eighteen weeks after starting on the oral multi-tyrosine kinase inhibitor TSU-68, laboratory investigations showed a substantial increase in serum transaminase levels (AST: 302 IU/l; ALT: 324 IU/l and an elevation of the HBV-DNA level (6.9 log copies/ml. The diagnosis was that the cause of the acute hepatitis was HBV reactivation and we immediately administered entecavir. Two months after the initiation of daily entecavir treatment, laboratory findings showed that the serum levels of transaminases and ALP had improved (AST: 18 IU/l; ALT: 10 IU/l; ALP: 197 U/l. When the HBV markers were examined 4 months later, they were altered: HBeAg was negative and HBeAb was positive. Entecavir treatment was discontinued after 6 months. Although reactivation with rituximab has been reported, reactivation with a tyrosine kinase inhibitor is extremely unusual in a patient who is HBsAg negative but anti-HBc positive. This is the first report describing HBV reactivation with an increasing HBV-DNA level in a HBsAg-negative/HBcAb-positive/HBsAb-positive patient who was treated with TSU-68 for hepatocellular carcinoma.

  18. Whole Genome Duplications Shaped the Receptor Tyrosine Kinase Repertoire of Jawed Vertebrates.

    Science.gov (United States)

    Brunet, Frédéric G; Volff, Jean-Nicolas; Schartl, Manfred

    2016-06-03

    The receptor tyrosine kinase (RTK) gene family, involved primarily in cell growth and differentiation, comprises proteins with a common enzymatic tyrosine kinase intracellular domain adjacent to a transmembrane region. The amino-terminal portion of RTKs is extracellular and made of different domains, the combination of which characterizes each of the 20 RTK subfamilies among mammals. We analyzed a total of 7,376 RTK sequences among 143 vertebrate species to provide here the first comprehensive census of the jawed vertebrate repertoire. We ascertained the 58 genes previously described in the human and mouse genomes and established their phylogenetic relationships. We also identified five additional RTKs amounting to a total of 63 genes in jawed vertebrates. We found that the vertebrate RTK gene family has been shaped by the two successive rounds of whole genome duplications (WGD) called 1R and 2R (1R/2R) that occurred at the base of the vertebrates. In addition, the Vegfr and Ephrin receptor subfamilies were expanded by single gene duplications. In teleost fish, 23 additional RTK genes have been retained after another expansion through the fish-specific third round (3R) of WGD. Several lineage-specific gene losses were observed. For instance, birds have lost three RTKs, and different genes are missing in several fish sublineages. The RTK gene family presents an unusual high gene retention rate from the vertebrate WGDs (58.75% after 1R/2R, 64.4% after 3R), resulting in an expansion that might be correlated with the evolution of complexity of vertebrate cellular communication and intracellular signaling.

  19. Spleen Tyrosine Kinase Regulates AP-1 Dependent Transcriptional Response to Minimally Oxidized LDL

    Science.gov (United States)

    Choi, Soo-Ho; Wiesner, Philipp; Almazan, Felicidad; Kim, Jungsu; Miller, Yury I.

    2012-01-01

    Oxidative modification of low-density lipoprotein (LDL) turns it into an endogenous ligand recognized by pattern-recognition receptors. We have demonstrated that minimally oxidized LDL (mmLDL) binds to CD14 and mediates TLR4/MD-2-dependent responses in macrophages, many of which are MyD88-independent. We have also demonstrated that the mmLDL activation leads to recruitment of spleen tyrosine kinase (Syk) to TLR4 and TLR4 and Syk phosphorylation. In this study, we produced a macrophage-specific Syk knockout mouse and used primary Syk−/− macrophages in our studies. We demonstrated that Syk mediated phosphorylation of ERK1/2 and JNK, which in turn phosphorylated c-Fos and c-Jun, respectively, as assessed by an in vitro kinase assay. c-Jun phosphorylation was also mediated by IKKε. c-Jun and c-Fos bound to consensus DNA sites and thereby completed an AP-1 transcriptional complex and induced expression of CXCL2 and IL-6. These results suggest that Syk plays a key role in TLR4-mediated macrophage responses to host-generated ligands, like mmLDL, with subsequent activation of an AP-1 transcription program. PMID:22384232

  20. Deletion of the endothelial Bmx tyrosine kinase decreases tumor angiogenesis and growth.

    Science.gov (United States)

    Holopainen, Tanja; López-Alpuche, Vanessa; Zheng, Wei; Heljasvaara, Ritva; Jones, Dennis; He, Yun; Tvorogov, Denis; D'Amico, Gabriela; Wiener, Zoltan; Andersson, Leif C; Pihlajaniemi, Taina; Min, Wang; Alitalo, Kari

    2012-07-15

    Bmx, [corrected] also known as Etk, is a member of the Tec family of nonreceptor tyrosine kinases. Bmx is expressed mainly in arterial endothelia and in myeloid hematopoietic cells. Bmx regulates ischemia-mediated arteriogenesis and lymphangiogenesis, but its role in tumor angiogenesis is not known. In this study, we characterized the function of Bmx in tumor growth using both Bmx knockout and transgenic mice. Isogenic colon, lung, and melanoma tumor xenotransplants showed reductions in growth and tumor angiogenesis in Bmx gene-deleted ((-/-)) mice, whereas developmental angiogenesis was not affected. In addition, growth of transgenic pancreatic islet carcinomas and intestinal adenomas was also slower in Bmx(-/-) mice. Knockout mice showed high levels of Bmx expression in endothelial cells of tumor-associated and peritumoral arteries. Moreover, endothelial cells lacking Bmx showed impaired phosphorylation of extracellular signal-regulated kinase (Erk) upon VEGF stimulation, indicating that Bmx contributes to the transduction of vascular endothelial growth factor signals. In transgenic mice overexpressing Bmx in epidermal keratinocytes, tumors induced by a two-stage chemical skin carcinogenesis treatment showed increased growth and angiogenesis. Our findings therefore indicate that Bmx activity contributes to tumor angiogenesis and growth.

  1. Imipramine protects retinal ganglion cells from oxidative stress through the tyrosine kinase receptor B signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Ming-lei Han; Guo-hua Liu; Jin Guo; Shu-juan Yu; Jing Huang

    2016-01-01

    Retinal ganglion cell (RGC) degeneration is irreversible in glaucoma and tyrosine kinase receptor B (TrkB)-associated signaling pathways have been implicated in the process. In this study, we attempted to examine whether imipramine, a tricyclic antidepressant, may protect hydrogen peroxide (H2O2)-induced RGC degeneration through the activation of the TrkB pathway in RGC-5 cell lines. RGC-5 cell lines were pre-treated with imipramine 30 minutes before exposure to H2O2. Western blot assay showed that in H2O2-damaged RGC-5 cells, imipramine activated TrkB pathways through extracellular signal-regulated protein kinase/TrkB phosphorylation. TUNEL staining assay also demonstrated that imipramine ameliorated H2O2-induced apoptosis in RGC-5 cells. Finally, TrkB-IgG intervention was able to reverse the protective effect of imipramine on H2O2-induced RGC-5 apoptosis. Imipramine therefore protects RGCs from oxidative stress-induced apoptosis through the TrkB signaling pathway.

  2. Imipramine protects retinal ganglion cells from oxidative stress through the tyrosine kinase receptor B signaling pathway

    Directory of Open Access Journals (Sweden)

    Ming-lei Han

    2016-01-01

    Full Text Available Retinal ganglion cell (RGC degeneration is irreversible in glaucoma and tyrosine kinase receptor B (TrkB-associated signaling pathways have been implicated in the process. In this study, we attempted to examine whether imipramine, a tricyclic antidepressant, may protect hydrogen peroxide (H 2 O 2 -induced RGC degeneration through the activation of the TrkB pathway in RGC-5 cell lines. RGC-5 cell lines were pre-treated with imipramine 30 minutes before exposure to H 2 O 2 . Western blot assay showed that in H 2 O 2 -damaged RGC-5 cells, imipramine activated TrkB pathways through extracellular signal-regulated protein kinase/TrkB phosphorylation. TUNEL staining assay also demonstrated that imipramine ameliorated H 2 O 2 -induced apoptosis in RGC-5 cells. Finally, TrkB-IgG intervention was able to reverse the protective effect of imipramine on H 2 O 2 -induced RGC-5 apoptosis. Imipramine therefore protects RGCs from oxidative stress-induced apoptosis through the TrkB signaling pathway.

  3. The tyrosine kinase Stitcher activates Grainy head and epidermal wound healing in Drosophila.

    Science.gov (United States)

    Wang, Shenqiu; Tsarouhas, Vasilios; Xylourgidis, Nikos; Sabri, Nafiseh; Tiklová, Katarína; Nautiyal, Naumi; Gallio, Marco; Samakovlis, Christos

    2009-07-01

    Epidermal injury initiates a cascade of inflammation, epithelial remodelling and integument repair at wound sites. The regeneration of the extracellular barrier and damaged tissue repair rely on the precise orchestration of epithelial responses triggered by the injury. Grainy head (Grh) transcription factors induce gene expression to crosslink the extracellular barrier in wounded flies and mice. However, the activation mechanisms and functions of Grh factors in re-epithelialization remain unknown. Here we identify stitcher (stit), a new Grh target in Drosophila melanogaster. stit encodes a Ret-family receptor tyrosine kinase required for efficient epidermal wound healing. Live imaging analysis reveals that Stit promotes actin cable assembly during wound re-epithelialization. Stit activation also induces extracellular signal-regulated kinase (ERK) phosphorylation along with the Grh-dependent expression of stit and barrier repair genes at the wound sites. The transcriptional stimulation of stit on injury triggers a positive feedback loop increasing the magnitude of epithelial responses. Thus, Stit activation upon wounding coordinates cytoskeletal rearrangements and the level of Grh-mediated transcriptional wound responses.

  4. Fyn tyrosine kinase regulates oligodendroglial cell development but is not required for morphological differentiation of oligodendrocytes.

    Science.gov (United States)

    Sperber, B R; McMorris, F A

    2001-02-15

    The non-receptor protein tyrosine kinase Fyn, which is a member of the Src family of kinases, has been shown to be essential for normal myelination and has been suggested to play a role in oligodendrocyte development. However, oligodendrocyte development has not been studied directly in cells lacking Fyn. Additionally, because Fyn is expressed in neurons as well as oligodendrocytes, it is possible that normal myelination requires Fyn expression in neurons but not in oligodendrocytes. To address these issues, we analyzed the development of oligodendrocytes in neuron-free glial cell cultures from fyn(-/-) mice that express no Fyn protein. We observed that oligodendrocytes develop to the stage where they elaborate an extensive network of membranous processes and express the antigenic components of mature oligodendrocytes in the complete absence of Fyn. However, as compared with fyn(+/+) controls, fewer oligodendroglia developed in fyn(-/-) cell cultures, and a smaller proportion of them matured to the stage characterized by a high degree of morphological complexity. In addition, we found that insulin-like growth factor-I, a potent stimulator of oligodendrocyte development, failed to stimulate morphological maturation of fyn(-/-) oligodendroglia. The pyrazolopyrimidine PP2, believed to be a selective inhibitor of Fyn, did not prevent the development of morphologically complex oligodendrocytes. Unexpectedly, however, it was toxic to both fyn(+/+) and fyn(-/-) glial cells, indicating that this class of inhibitors can have significant effects that are independent of Fyn.

  5. Dialkoxyquinazolines: Screening Epidermal Growth Factor ReceptorTyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

    Energy Technology Data Exchange (ETDEWEB)

    VanBrocklin, Henry F.; Lim, John K.; Coffing, Stephanie L.; Hom,Darren L.; Negash, Kitaw; Ono, Michele Y.; Hanrahan, Stephen M.; Taylor,Scott E.; Vanderpoel, Jennifer L.; Slavik, Sarah M.; Morris, Andrew B.; Riese II, David J.

    2005-09-01

    The epidermal growth factor receptor (EGFR), a long-standingdrug development target, is also a desirable target for imaging. Sixteendialkoxyquinazoline analogs, suitable for labeling with positron-emittingisotopes, have been synthesized and evaluated in a battery of in vitroassays to ascertain their chemical and biological properties. Thesecharacteristics provided the basis for the adoption of a selection schemato identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of thecompounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFRtyrosine kinase. All of the analogs inhibited ligand-induced EGFRtyrosine phosphorylation (IC50 = 0.8 - 20 nM). The HPLC-estimatedoctanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline aswell as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the bestcombination of characteristics that warrant radioisotope labeling andfurther evaluation in tumor-bearing mice.

  6. Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition.

    Science.gov (United States)

    Caffa, Irene; D'Agostino, Vito; Damonte, Patrizia; Soncini, Debora; Cea, Michele; Monacelli, Fiammetta; Odetti, Patrizio; Ballestrero, Alberto; Provenzani, Alessandro; Longo, Valter D; Nencioni, Alessio

    2015-05-20

    Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use.

  7. Spleen tyrosine kinase (SYK) is a potential target for the treatment of cutaneous lupus erythematosus patients.

    Science.gov (United States)

    Braegelmann, Christine; Hölzel, Michael; Ludbrook, Valerie; Dickson, Marion; Turan, Nil; Ferring-Schmitt, Sandra; Sternberg, Sonja; Bieber, Thomas; Kuhn, Annegret; Wenzel, Joerg

    2016-05-01

    Spleen tyrosine kinase (SYK) is a protein kinase involved in cell proliferation and the regulation of inflammatory pathways. Due to the increasing evidence that kinase inhibitors have potential as specific anti-inflammatory drugs, we have investigated the potential for SYK inhibition as a therapeutic target in autoimmune diseases, particularly cutaneous lupus erythematosus (CLE). Skin samples of patients with different CLE subtypes and appropriate controls were analysed for the expression of SYK and SYK-associated pro-inflammatory mediators via gene expression analysis and immunohistochemistry. The functional role of SYK in keratinocytes was investigated in vitro, using LE-typical pro-inflammatory stimuli and a selective inhibitor of SYK. SYK-associated genes are strongly upregulated in CLE skin lesions. Importantly, phosphorylated SYK (pSYK) is strongly expressed by several immune cell types and also keratinocytes in CLE skin. In vitro, immunostimulatory nucleic acids are capable of inducing SYK phosphorylation in keratinocytes leading to the induction of pro-inflammatory cytokines, while small-molecule SYK inhibition decreases the expression of these proteins. The results demonstrate that pSYK is expressed by immune cells and keratinocytes in skin lesions of CLE patients. LE-typical stimuli induce the expression of pSYK in vitro. Small-molecule SYK inhibition leads to a reduction of pSYK expression and downregulation of pro-inflammatory cytokines in keratinocytes. We therefore believe that pSYK provides a potential future drug target for the treatment of patients who suffer from CLE and related skin disorders. Specifically, our study reveals evidence supporting the use of topical SYK inhibitors in treating lupus.

  8. The pathobiology of the oncogenic tyrosine kinase NPM-ALK: a brief update.

    Science.gov (United States)

    Lai, Raymond; Ingham, Robert J

    2013-04-01

    Extensive research has been carried out in the past two decades to study the pathobiology of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), which is an oncogenic fusion protein found exclusively in a specific type of T-cell lymphoid malignancy, namely ALK-positive anaplastic large cell lymphoma. Results from these studies have provided highly useful insights into the mechanisms by which a constitutively tyrosine kinase, such as NPM-ALK, promotes tumorigenesis. Several previous publications have comprehensively summarized the advances in this field. In this review, we provide readers with a brief update on specific areas of NPM-ALK pathobiology. In the first part, the NPM-ALK/signal transducer and activator of transcription 3 (STAT3) signaling axis is discussed, with an emphasis on the existence of multiple biochemical defects that have been shown to amplify the oncogenic effects of this signaling axis. Specifically, findings regarding JAK3, SHP1 and the stimulatory effects of several cytokines including interleukin (IL)-9, IL-21 and IL-22 are summarized. New concepts stemming from recent observations regarding the functional interactions among the NPM-ALK/STAT3 axis, β catenin and glycogen synthase kinase 3β will be postulated. Lastly, new mechanisms by which the NPM-ALK/STAT3 axis promotes tumorigenesis, such as its modulations of Twist1, hypoxia-induced factor 1α, CD274, will be described. In the second part, we summarize recent data generated by mass spectrometry studies of NPM-ALK, and use MSH2 and heat shock proteins as examples to illustrate the use of mass spectrometry data in stimulating new research in this field. In the third part, the evolving field of microRNA in the context of NPM-ALK biology is discussed.

  9. The tyrosine 343 residue of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions.

    Science.gov (United States)

    Hegazy, Samar A; Wang, Peng; Anand, Mona; Ingham, Robert J; Gelebart, Pascal; Lai, Raymond

    2010-06-25

    The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK(K210R) mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr(338), Tyr(342), and Tyr(343)) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr(338) or Tyr(342) did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr(343) abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK(Y343F) mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK(Y343F). In conclusion, we identified Tyr(343) of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.

  10. 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.

  11. Tyrosine kinase Etk/BMX protects nasopharyngeal carcinoma cells from apoptosis induced by radiation.

    Science.gov (United States)

    Zhang, Zhenhua; Zhu, Weiliang; Zhang, Jian; Guo, Linlang

    2011-04-01

    Etk (Epithelial and endothelial tyrosine kinase), also known as Bmx (bone marrow X kinase) plays an important role in apoptosis of cancer cells. The purpose of this study was to investigate whether Etk/Bmx is involved in the apoptosis induced by irradiation in NPC cells and correlated with the apoptosis associated proteins such as p53, Bcl-2, Bcl-X(L) and Bak. To this end, we first developed a NPC subline (SUNE1-Etk) by transfection. The SUNE1-Etk that over-expresses Etk/BMX and its parental SUNE1 cell line were used to confirm whether Etk/BMX can protect NPC cells from apoptosis induced by radiation. The proliferation rates or the level of cell survival following irradiation were assessed by MTT and flow cytometry. Tumorigenecity study was done to substantiate the results in vitro. The results showed that the cell viability was significantly higher in SUNE1-Etk cells than that in parental SUNE1 cells in vitro, and tumors inoculated with SUNE1-Etk cells grew rapidly than those with SUNE1 after irradiation treatment. Our data also demonstrated that the up-expression of Etk/BMX increased G(2)/M arrest in response to irradiation. The protein level of p53 was greatly down-regulated whereas Bcl-2 was up-regulated, after irradiation treatment of SUNE1-Etk cells. Our results suggested that Etk/BMX may play a role in protection of NPC cells from apoptosis, and both p53 and Bcl-2 may be involved in radiation-induced apoptosis through Etk/Bmx pathway in NPC cells.

  12. Axl receptor tyrosine kinase is a novel target of apigenin for the inhibition of cell proliferation.

    Science.gov (United States)

    Kim, Kyung-Chan; Choi, Eun-Ha; Lee, Chuhee

    2014-08-01

    The Axl receptor tyrosine kinase (RTK), along with Tyro 3 and Mer, belongs to the TAM subfamily that promotes survival, stimulates proliferation and/or inhibits apoptosis. In various types of human cancer, including breast, lung and prostate cancer, Axl expression is increased and correlates with an advanced clinical stage. In this study, we examined whether apigenin has an effect on Axl expression, which in turn can affect cell proliferation. The treatment of the non‑small cell lung cancer (NSCLC) cells, A549 and H460, with apigenin decreased Axl mRNA and protein expression in a dose‑dependent manner. Axl promoter activity was also inhibited by apigenin, indicating that apigenin suppressed Axl expression at the transcriptional level. Upon treatment with apigenin, the viability of both the A549 and H460 cells was gradually decreased and the anti-proliferative effects were further confirmed by the dose‑dependent decrease in the clonogenic ability of the apigenin‑treated cells. Subsequently, we found that the viability and clonogenic ability of the cells treated with apigenin was less or more affected by transfection of the cells with a Axl-expressing plasmid or Axl targeting siRNA, compared to transfection with the empty vector or control siRNA, respectively. In addition, apigenin increased the expression of p21, a cyclin-dependent kinase inhibitor, but reduced the expression of X-linked inhibitor of apoptosis protein (XIAP). These cell cycle arrest and pro-apoptotic effects of apigenin were also attenuated or augmented by the up- or downregulation of Axl expression, respectively, which suggests that Axl is a novel target of apigenin through which it exerts its inhibitory effects on cell proliferation. Taken together, our data indicate that apigenin downregulates Axl expression, which subsequently results in the inhibition of NSCLC cell proliferation through the increase and decrease of p21 and XIAP expression, respectively.

  13. The Plasticity of Oncogene Addiction: Implications for Targeted Therapies Directed to Receptor Tyrosine Kinases

    Directory of Open Access Journals (Sweden)

    Vinochani Pillay

    2009-05-01

    Full Text Available A common mutation of the epidermal growth factor receptor (EGFR in glioblastoma multiforme (GBM is an extracellular truncation known as the de2-7 EGFR (or EGFRvIII. Hepatocyte growth factor (HGF is the ligand for the receptor tyrosine kinase (RTK c-Met, and this signaling axis is often active in GBM. The expression of the HGF/c-Met axis or de2-7 EGFR independently enhances GBMgrowth and invasiveness, particularly through the phosphatidylinositol-3 kinase/pAkt pathway. Using RTK arrays, we show that expression of de2-7 EGFR in U87MG GBM cells leads to the coactivation of several RTKs, including platelet-derived growth factor receptor β and c-Met. A neutralizing antibody to HGF (AMG102 did not inhibit de2-7 EGFR-mediated activation of c-Met, demonstrating that it is ligand-independent. Therapy for parental U87MG xenografts with AMG 102 resulted in significant inhibition of tumor growth, whereas U87MG.Δ2-7 xenografts were profoundly resistant. Treatment of U87MG.Δ2-7 xenografts with panitumumab, an anti-EGFR antibody, only partially inhibited tumor growth as xenografts rapidly reverted to the HGF/c-Met signaling pathway. Cotreatment with panitumumab and AMG 102 prevented this escape leading to significant tumor inhibition through an apoptotic mechanism, consistent with the induction of oncogenic shock. This observation provides a rationale for using panitumumab and AMG 102 in combination for the treatment of GBM patients. These results illustrate that GBM cells can rapidly change the RTK driving their oncogene addiction if the alternate RTK signals through the same downstream pathway. Consequently, inhibition of a dominant oncogene by targeted therapy can alter the hierarchy of RTKs resulting in rapid therapeutic resistance.

  14. 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....

  15. A platinum-based hybrid drug design approach to circumvent acquired resistance to molecular targeted tyrosine kinase inhibitors

    Science.gov (United States)

    Wei, Yuming; Poon, Daniel C.; Fei, Rong; Lam, Amy S. M.; Au-Yeung, Steve C. F.; To, Kenneth K. W.

    2016-05-01

    Three molecular targeted tyrosine kinase inhibitors (TKI) were conjugated to classical platinum-based drugs with an aim to circumvent TKI resistance, predominately mediated by the emergence of secondary mutations on oncogenic kinases. The hybrids were found to maintain specificity towards the same oncogenic kinases as the original TKI. Importantly, they are remarkably less affected by TKI resistance, presumably due to their unique structure and the observed dual mechanism of anticancer activity (kinase inhibition and DNA damage). The study is also the first to report the application of a hybrid drug approach to switch TKIs from being efflux transporter substrates into non-substrates. TKIs cannot penetrate into the brain for treating metastases because of efflux transporters at the blood brain barrier. The hybrids were found to escape drug efflux and they accumulate more than the original TKI in the brain in BALB/c mice. Further development of the hybrid compounds is warranted.

  16. A platinum-based hybrid drug design approach to circumvent acquired resistance to molecular targeted tyrosine kinase inhibitors.

    Science.gov (United States)

    Wei, Yuming; Poon, Daniel C; Fei, Rong; Lam, Amy S M; Au-Yeung, Steve C F; To, Kenneth K W

    2016-05-06

    Three molecular targeted tyrosine kinase inhibitors (TKI) were conjugated to classical platinum-based drugs with an aim to circumvent TKI resistance, predominately mediated by the emergence of secondary mutations on oncogenic kinases. The hybrids were found to maintain specificity towards the same oncogenic kinases as the original TKI. Importantly, they are remarkably less affected by TKI resistance, presumably due to their unique structure and the observed dual mechanism of anticancer activity (kinase inhibition and DNA damage). The study is also the first to report the application of a hybrid drug approach to switch TKIs from being efflux transporter substrates into non-substrates. TKIs cannot penetrate into the brain for treating metastases because of efflux transporters at the blood brain barrier. The hybrids were found to escape drug efflux and they accumulate more than the original TKI in the brain in BALB/c mice. Further development of the hybrid compounds is warranted.

  17. Anti-Angiogenic Therapy: Strategies to Develop Potent VEGFR-2 Tyrosine Kinase Inhibitors and Future Prospect.

    Science.gov (United States)

    Shi, Leilei; Zhou, Jianfeng; Wu, Jifeng; Shen, Yuemao; Li, Xun

    2016-01-01

    Tumor angiogenesis has always been a major gap for effective cancer therapy. Interruption of aberrant angiogenesis by specific inhibitors targeting receptor tyrosine kinases (RTKs) has been of great interests to medicinal chemists. Among the factors that are involved in tumor angiogenesis, vascular endothelial growth factor receptor-2 (VEGFR-2) is validated as the most closely related factor which can drive angiogenesis through binding with its natural ligand VEGF. The well-validated VEGF-driven VEGFR-2 signaling pathway can stimulate many endothelial responses, including increasing vessel permeability and enhancing endothelial cell proliferation, migration and differentiation. Consequently, circumventing angiogenesis by VEGFR-2 inhibitors represents a promising strategy for counteracting various VEGFR-2-mediated disorders as well as drug resistance. Over the past decades, a considerable number of novel small molecular VEGFR-2 inhibitors have been exploited with diverse chemical scaffolds. Especially, recent frequently launched inhibitors have declared their research values and therapeutic potentials in oncology. Still, the antiangiogenesis based treatment remains an ongoing challenge. In this review, a comprehensive retrospective of newly emerged VEGFR-2 inhibitors have been summarized, with the emphasis on the structure-activity relationship (SAR) investigation, and also binding patterns of representative inhibitors with biotargets. On the basis of all of this information, varied strategies for developing potent VEGFR-2 inhibitors and the future prospect of the clinical application of antiangiogenic inhibitors are discussed hereby.

  18. Induction of human pancreatic beta cell replication by inhibitors of dual specificity tyrosine regulated kinase

    Science.gov (United States)

    Wang, Peng; Alvarez-Perez, Juan-Carlos; Felsenfeld, Dan P.; Liu, Hongtao; Sivendran, Sharmila; Bender, Aaron; Kumar, Anil; Sanchez, Roberto; Scott, Donald K.; Garcia-Ocaña, Adolfo; Stewart, Andrew F.

    2015-01-01

    Types 1 and 2 diabetes affect some 380 million people worldwide. Both result ultimately from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with peak beta cell labeling indices achieving approximately 2% in first year of life1-4. In embryonic life and after early childhood, beta cell replication rates are very low. While beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts1-8. Hence, there remains an urgent need for diabetes therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, we report the results of a high-throughput small molecule screen (HTS) revealing a novel class of human beta cell mitogenic compounds, analogues of the small molecule, harmine. We also define dual specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine, and the Nuclear Factors of activated T-cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation as well as beta cell differentiation. These observations suggest that harmine analogues (“harmalogs”) may have unique therapeutic promise for human diabetes therapy. Enhancing potency and beta cell specificity are important future challenges. PMID:25751815

  19. Mediation of transitional B cell maturation in the absence of functional Bruton’s tyrosine kinase

    Science.gov (United States)

    Tanwar, Shalini; Dhar, Atika; Varanasi, Vineeth; Mukherjee, Tapas; Boppana, Ramanamurthy; Basak, Soumen; Bal, Vineeta; George, Anna; Rath, Satyajit

    2017-01-01

    X-linked immune-deficient (Xid) mice, carrying a mutation in Bruton’s tyrosine kinase (Btk), have multiple B cell lineage differentiation defects. We now show that, while Xid mice showed only mild reduction in the frequency of the late transitional (T2) stage of peripheral B cells, the defect became severe when the Xid genotype was combined with either a CD40-null, a TCRbeta-null or an MHC class II (MHCII)-null genotype. Purified Xid T1 and T2 B cells survived poorly in vitro compared to wild-type (WT) cells. BAFF rescued WT but not Xid T1 and T2 B cells from death in culture, while CD40 ligation equivalently rescued both. Xid transitional B cells ex vivo showed low levels of the p100 protein substrate for non-canonical NF-kappaB signalling. In vitro, CD40 ligation induced equivalent activation of the canonical but not of the non-canonical NF-kappaB pathway in Xid and WT T1 and T2 B cells. CD40 ligation efficiently rescued p100-null T1 B cells from neglect-induced death in vitro. These data indicate that CD40-mediated signals, likely from CD4 T cells, can mediate peripheral transitional B cell maturation independent of Btk and the non-canonical NF-kappaB pathway, and thus contribute to the understanding of the complexities of peripheral B cell maturation. PMID:28378771

  20. The Tyrosine Kinase Inhibitor Sunitinib Affects Ovulation but Not Ovarian Reserve in Mouse: A Preclinical Study

    Science.gov (United States)

    Bernard, Valérie; Bouilly, Justine; Kramer, Piet; Carré, Nadège; Schlumberger, Martin; Visser, Jenny A.; Young, Jacques; Binart, Nadine

    2016-01-01

    The aim of the study was to evaluate ovarian toxicity of tyrosine kinase inhibitor (TKI) sunitinib, since only scarce data are available on gonadal function after this treatment. Six-week-old female mice received orally, once daily, vehicle or sunitinib (50 mg/kg/d) during 5 weeks. Fertility parameters were analyzed from ovulation to litter assessment. Sunitinib exposure significantly reduced (i) corpora lutea number per ovary (1.1 ± 0.38 in sunitinib group versus 4 ± 0.79 in control group, p<0.01) and (ii) serum Anti Müllerian hormone (AMH) levels in sunitinib treated mice (12.01 ± 1.16) compared to control mice (14.33 ± 0.87 ng/ml, p< 0.05). However, primordial and growing follicles numbers per ovary were not different in both groups. After treatment withdrawal, female mice in both groups were able to obtain litters. These data could be helpful to counsel clinicians and patients, when fertility preservation methods are discussed, before TKI treatment in girls and young women. PMID:27035144

  1. Mice lacking Axl and Mer tyrosine kinase receptors are susceptible to experimental autoimmune orchitis induction.

    Science.gov (United States)

    Li, Nan; Liu, Zhenghui; Zhang, Yue; Chen, Qiaoyuan; Liu, Peng; Cheng, C Yan; Lee, Will M; Chen, Yongmei; Han, Daishu

    2015-03-01

    The mammalian testis is an immunoprivileged organ where male germ cell autoantigens are immunologically ignored. Both systemic immune tolerance to autoantigens and local immunosuppressive milieu contribute to the testicular immune privilege. Testicular immunosuppression has been intensively studied, but information on systemic immune tolerance to autoantigens is lacking. In the present study, we aimed to determine the role of Axl and Mer receptor tyrosine kinases in maintaining the systemic tolerance to male germ cell antigens using the experimental autoimmune orchitis (EAO) model. Axl and Mer double-knockout (Axl(-/-)Mer(-/-)) mice developed evident EAO after a single immunization with germ cell homogenates emulsified with complete Freund's adjuvant. EAO was characterized by the accumulation of macrophages and T lymphocytes in the testis. Damage to the seminiferous epithelium was also observed. EAO induction was associated with pro-inflammatory cytokine upregulation in the testes, impaired permeability of the blood-testis barrier and generation of autoantibodies against germ cell antigens in Axl(-/-)Mer(-/-) mice. Immunization also induced mild EAO in Axl or Mer single-gene-knockout mice. By contrast, a single immunization failed to induce EAO in wild-type mice. The results indicate that Axl and Mer receptors cooperatively regulate the systemic immune tolerance to male germ cell antigens.

  2. Lenalidomide in chronic lymphocytic leukemia: the present and future in the era of tyrosine kinase inhibitors.

    Science.gov (United States)

    Maffei, Rossana; Colaci, Elisabetta; Fiorcari, Stefania; Martinelli, Silvia; Potenza, Leonardo; Luppi, Mario; Marasca, Roberto

    2016-01-01

    Lenalidomide is an immunomodulatory agent (IMiD) clinically active in chronic lymphocytic leukemia (CLL), both in heavily pre-treated patients and upfront. Lenalidomide has a unique mechanism of action in CLL. Its efficacy relies on a multifactorial mode-of-action (MOA), comprising a plethora of immunomodulatory actions, the disruption of mutualistic interactions inside CLL microenvironment and direct effects against leukemic cells. In the last few years, a number of new and highly effective drugs appeared in the scenario of CLL therapeutic options, i.e. tyrosine kinase inhibitors (TKIs), showing a good safety profile and impressive clinical response, also in high-risk patients. In this review, we describe the data from clinical studies about lenalidomide efficacy in CLL and we critically dissect the different mechanisms of action of this drug. We point the attention on open issues, including drug dosage and administration schedule, prediction of clinical response to lenalidomide, and combination therapeutic strategies. This overview would be useful to envision a possible role of lenalidomide in the treatment flow-chart of CLL, exploiting its peculiar MOA and also exploring the possible synergetic effect with new drugs.

  3. Third generation tyrosine kinase inhibitors and their development in advanced renal cell carcinoma.

    Science.gov (United States)

    Bukowski, Ronald M

    2012-01-01

    Angiogenesis in general and the vascular endothelial growth factor (VEGF) signaling axis in particular is a validated target in renal cell carcinoma (RCC). Clear-cell carcinoma of the kidney is now recognized as a malignancy that is sensitive to inhibitors of the VEGF pathway. Treatment options for patients with metastatic renal cell carcinoma have evolved in dramatic fashion over the past 6 years, and a new paradigm has developed. The cytokines interferon-α and interleukin-2 were previously utilized for therapy, but since December 2005, six new agents have been approved in the United States for the treatment of advanced RCC. Two are tyrosine kinase inhibitors (TKI's) including sunitinib and recently pazopanib, and the multikinase inhibitor sorafenib. The current review examines the evolving data with the next generation of TKI's, axitinib and tivozanib being developed for the treatment of advanced RCC. These agents were synthesized to provide increased target specificity and enhanced target inhibition. The preclinical and clinical data are examined, an overview of the development of these TKI's is provided, and discussion plus speculation concerning their potential roles as RCC therapy is provided.

  4. Review : Third Generation Tyrosine Kinase Inhibitors and Their Development in Advanced Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Ronald M Bukowski

    2012-02-01

    Full Text Available Angiogenesis in general and the VEGF signaling axis in particular is a validated target in renal cell carcinoma. Clear cell carcinoma of the kidney is now recognized as a malignancy that is sensitive to inhibitors of the vascular endothelial growth factor pathway. Treatment options for patients with metastatic renal cell carcinoma have evolved in dramatic fashion over the past six years, and a new paradigm has developed. The cytokines interferon-α and interleukin-2 were previously utilized for therapy, but since December 2005, six new agents have been approved in the United States for the treatment of advanced RCC. Three are tyrosine kinase inhibitors (TKI’s including sunitinib, sorafenib, and recently pazopanib. The current review examines the evolving data with the next generation of TKI’s, axitinib and tivozanib being developed for the treatment of advanced RCC. These agents were synthesized to provide increased target specificity and enhanced target inhibition. The preclinical and clinical data are examined, an overview of the development of these TKI’s is provided, and discussion plus speculation concerning their potential roles as RCC therapy is provided.

  5. Overexpression of Fyn tyrosine kinase causes abnormal development of primary sensory neurons in Xenopus laevis embryos.

    Science.gov (United States)

    Saito, R; Fujita, N; Nagata, S

    2001-06-01

    The expression and function of the Src family protein tyrosine kinase Fyn in Xenopus laevis embryos have been examined. In situ hybridization analysis demonstrated nervous system-specific expression of Fyn mRNA in tail-bud embryos. However, a class of primary sensory neurons; that is, Rohon-Beard (RB) neurons, which is positive for immunoglobulin superfamily cell adhesion molecules (CAM), neural cell adhesion molecule (N-CAM) and contactin, is devoid of Fyn expression. Injection of Fyn mRNA into one of the blastomeres at the 2-cell stage led to overexpression of Fyn in the injected half of the tail-bud embryos. Immunolabeling of the embryos with anti-HNK-1 antibody revealed that the peripheral axons of RB neurons were partially misguided and bound to each other to form abnormal subcutaneous fascicles. Similar abnormality was induced by injection of the Fyn overexpression vector. The incidence of abnormality appeared dose-dependent, being 68-92% of the injected embryos at 50-400 pg of mRNA. Co-injection of the contactin antisense vector depleted contactin mRNA accumulation without affecting Fyn overexpression and reduced the incidence of the abnormal RB-cell phenotype. However, the N-CAM antisense was ineffective in reducing this abnormality. These results suggest that Fyn can modify signals regulating axonal guidance or fasciculation in the developing X. laevis nervous system and that contactin may affect this action of Fyn.

  6. Phosphorylation impact on Spleen Tyrosine kinase conformation by Surface Enhanced Raman Spectroscopy

    Science.gov (United States)

    Cottat, Maximilien; Yasukuni, Ryohei; Homma, Yo; Lidgi-Guigui, Nathalie; Varin-Blank, Nadine; Lamy de La Chapelle, Marc; Le Roy, Christine

    2017-01-01

    Spleen Tyrosine Kinase (Syk) plays a crucial role in immune cell signalling and its altered expression or activation are involved in several cancers. Syk activity relies on its phosphorylation status and its multiple phosphorylation sites predict several Syk conformations. In this report, we characterized Syk structural changes according to its phosphorylation/activation status by Surface Enhanced Raman Spectroscopy (SERS). Unphosphorylated/inactive and phosphorylated/active Syk forms were produced into two expression systems with different phosphorylation capability. Syk forms were then analysed by SERS that was carried out in liquid condition on a lithographically designed gold nanocylinders array. Our study demonstrated that SERS signatures of the two Syk forms were drastically distinct, indicating structural modifications related to their phosphorylation status. By comparison with the atomic structure of the unphosphorylated Syk, the SERS peak assignments of the phosphorylated Syk nearest gold nanostructures revealed a differential interaction with the gold surface. We finally described a model for Syk conformational variations according to its phosphorylation status. In conclusion, SERS is an efficient technical approach for studying in vitro protein conformational changes and might be a powerful tool to determine protein functions in tumour cells.

  7. The Dtk receptor tyrosine kinase, which binds protein S, is expressed during hematopoiesis.

    Science.gov (United States)

    Crosier, P S; Freeman, S A; Orlic, D; Bodine, D M; Crosier, K E

    1996-02-01

    Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.

  8. Pilose antler polypeptides promote chondrocyte proliferation via the tyrosine kinase signaling pathway

    Directory of Open Access Journals (Sweden)

    Lin Jian-Hua

    2011-11-01

    Full Text Available Abstract Background Pilose antler polypeptides (PAP have been reported to promote chondrocyte proliferation. However, the underlying mechanism remains unclear. The present study was to investigate the effects of PAP on the proliferation of chondrocytes and its underlying mechanism. Methods Chondrocytes isolated from the knee of Zealand white rabbits were cultured. The second generation chondrocytes were collected and identified using safranin-O staining. The chondrocytes were divided into the following 4 groups including serum-free, PAP, genistein (an inhibitor of tyrosine kinases, and PAP plus genistein group. Cell viability was analyzed using the MTT assay. The cell cycle distribution of the chondrocytes was analyzed by flow cytometry. The expression levels of cyclin A was detected using immunocytochemical staining. Results No significant difference was observed between serum-free and genistein group. Treatment of the cultures with PAP produced a significant dose-dependent increase in cell viability, the percentage proportion of chondrocytes in the S phase and Cyclin A expression as well. However, the promoting effect of PAP on chondrocyte proliferation were dose-dependently inhibited by genistein, whereas genistein alone had no effect on proliferation of isolated chondrocytes. Conclusions The data demonstrate that PAP promotes chondrocyte proliferation with the increased cell number, percentage proportion of chondrocytes in S phase and expression of protein cyclin A via the TK signaling pathway.

  9. Tyrosine kinase domain mutations of EGFR gene in head and neck squamous cell carcinoma

    Science.gov (United States)

    Vatte, Chittibabu; Al Amri, Ali M; Cyrus, Cyril; Chathoth, Shahanas; Acharya, Sadananda; Hashim, Tariq Mohammad; Al Ali, Zhara; Alshreadah, Saleh Tawfeeq; Alsayyah, Ahmed; Al-Ali, Amein K

    2017-01-01

    Background Epidermal growth factor receptor (EGFR) is a commonly altered gene that is identified in various cancers, including head and neck squamous cell carcinoma (HNSCC). Therefore, EGFR is a promising molecular marker targeted by monoclonal antibodies and small molecule inhibitors targeting the tyrosine kinase (TK) domain. Objective The objective of this study was to investigate the spectrum of mutations in exons 18, 19, 20, and 21 of the EGFR gene in HNSCC patients. Materials and methods This retrospective study included 47 confirmed HNSCC cases. Mutations in the TK domain, exons 18, 19, 20, and 21 of the EGFR gene, were detected by Scorpion® chemistry and ARMS® technologies on Rotor-Gene Q real-time polymerase chain reaction. Results The tumors exhibited EGFR-TK domain mutations in 57% of cases. Four cases of T790M mutations were reported for the first time among HNSCC patients. Out of the total mutations, L861Q (exon 21), exon 20 insertions and deletions of exon 19 accounted for the majority of mutations (21%, 19%, and 17%, respectively). EGFR mutation status was correlated with the higher grade (P=0.026) and advanced stage (P=0.034) of HNSCC tumors. Conclusion Higher frequency of EGFR-TK domain mutations together with the presence of the T790M mutation suggests that identification of these mutations might streamline the therapy and provide a better prognosis in HNSCC cases. PMID:28352186

  10. The Tyrosine Kinase Inhibitor Sunitinib Affects Ovulation but Not Ovarian Reserve in Mouse: A Preclinical Study.

    Directory of Open Access Journals (Sweden)

    Valérie Bernard

    Full Text Available The aim of the study was to evaluate ovarian toxicity of tyrosine kinase inhibitor (TKI sunitinib, since only scarce data are available on gonadal function after this treatment. Six-week-old female mice received orally, once daily, vehicle or sunitinib (50 mg/kg/d during 5 weeks. Fertility parameters were analyzed from ovulation to litter assessment. Sunitinib exposure significantly reduced (i corpora lutea number per ovary (1.1 ± 0.38 in sunitinib group versus 4 ± 0.79 in control group, p<0.01 and (ii serum Anti Müllerian hormone (AMH levels in sunitinib treated mice (12.01 ± 1.16 compared to control mice (14.33 ± 0.87 ng/ml, p< 0.05. However, primordial and growing follicles numbers per ovary were not different in both groups. After treatment withdrawal, female mice in both groups were able to obtain litters. These data could be helpful to counsel clinicians and patients, when fertility preservation methods are discussed, before TKI treatment in girls and young women.

  11. Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2.

    Science.gov (United States)

    Chandler, Kevin Brown; Leon, Deborah R; Meyer, Rosana D; Rahimi, Nader; Costello, Catherine E

    2017-02-03

    Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important receptor tyrosine kinase (RTK) that plays critical roles in both physiologic and pathologic angiogenesis. The extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites (sequons). N-glycosylation plays a central role in RTK ligand binding, trafficking, and stability. However, despite its importance, the functional role of N-glycosylation of VEGFR-2 remains poorly understood. The objectives of the present study were to characterize N-glycosylation sites in VEGFR-2 via enzymatic release of the glycans and concomitant incorporation of (18)O into formerly N-glycosylated sites followed by tandem mass spectrometry (MS/MS) analysis to determine N-glycosylation site occupancy and the site-specific N-glycan heterogeneity of VEGFR-2 glycopeptides. The data demonstrated that all seven VEGFR-2 immunoglobulin-like domains have at least one occupied N-glycosylation site. MS/MS analyses of glycopeptides and deamidated, deglycosylated (PNGase F-treated) peptides from ectopically expressed VEGFR-2 in porcine aortic endothelial (PAE) cells identified N-glycans at the majority of the 17 potential N-glycosylation sites on VEGFR-2 in a site-specific manner. The data presented here provide direct evidence for site-specific, heterogeneous N-glycosylation and N-glycosylation site occupancy on VEGFR-2. The study has important implications for the therapeutic targeting of VEGFR-2, ligand binding, trafficking, and signaling.

  12. Extracellular domain determinants of LET-23 (EGF) receptor tyrosine kinase activity in Caenorhabditis elegans.

    Science.gov (United States)

    Moghal, Nadeem; Sternberg, Paul W

    2003-08-21

    Negative regulation of ErbB/EGFR signalling pathways is important for normal development and the prevention of cancer. In a genetic screen to uncover mechanisms that negatively regulate ErbB signalling in Caenorhabditis elegans, we isolated a second-site mutation (sy621) that promotes the activity of a gain-of-function allele (sa62gf) of the let-23 (EGF) receptor tyrosine kinase. We show that activation by the sa62 mutation (C359Y) likely results from a break in the conserved disulphide-bonded eighth module at the junction of CR1 and L2. The sy621 mutation causes a G270E change in the third disulphide-bonded module of CR1, and causes no phenotype on its own, but cooperates with the sa62 mutation to promote receptor activity. Although both sa62 single- and double-mutant receptors can function in the absence of ligand, they can be further activated by ligand. Our results support the current model for ligand-induced dimerization based on the recent crystal structures of HER3 and the EGFR, and provide more evidence for the generation of distinctly activated ErbB family members through mutation.

  13. Dermatologic Toxicities from Monoclonal Antibodies and Tyrosine Kinase Inhibitors against EGFR: Pathophysiology and Management

    Directory of Open Access Journals (Sweden)

    Shaad E. Abdullah

    2012-01-01

    Full Text Available Epidermal growth factor receptor (EGFR inhibition has now been well established as an effective treatment for various cancers. The EGFR belongs to the ErbB family of tyrosine kinase receptors which regulate tumor cell differentiation, survival and proliferation. Activation of EGFR drives tumorigenesis in lung, head and neck, colorectal and pancreatic cancers. Irrespective of the type of cancer being treated and the mechanism by which tumor EGFR drives tumorigenesis, the major side effect of EGFR inhibition is a papulopustular (also described as maculopapular or acneiform rash which occurs in about two thirds of treated patients. Interestingly, this rash has been commonly correlated with better clinical outcomes (objective tumor response and patient survival. The pathophysiology of dermatological toxicity from EGFR inhibitors is an important area of clinical research, and the proper management of the rash is essential to increase the therapeutic index from this class of drugs. In this paper, we review the dermatologic toxicities associated with EGFR inhibitors with an emphasis on its pathophysiology and clinical management.

  14. UV-Vis spectroscopy and solvatochromism of the tyrosine kinase inhibitor AG-1478.

    Science.gov (United States)

    Khattab, Muhammad; Wang, Feng; Clayton, Andrew H A

    2016-07-05

    The effect of twenty-one solvents on the UV-Vis spectrum of the tyrosine kinase inhibitor AG-1478 was investigated. The absorption spectrum in the range 300-360nm consisted of two partially overlapping bands at approximately 340nm and 330nm. The higher energy absorption band was more sensitive to solvent and exhibited a peak position that varied from 327nm to 336nm, while the lower energy absorption band demonstrated a change in peak position from 340nm to 346nm in non-chlorinated solvents. The fluorescence spectrum of AG-1478 was particularly sensitive to solvent. The wavelength of peak intensity varied from 409nm to 495nm with the corresponding Stokes shift in the range of 64nm to 155nm (4536cm(-1) to 9210cm(-1)). We used a number of methods to assess the relationship between spectroscopic properties and solvent properties. The detailed analysis revealed that for aprotic solvents, the peak position of the emission spectrum in wavenumber scale correlated with the polarity (dielectric constant or ET(30)) of the solvent. In protic solvents, a better correlation was observed between the hydrogen bonding power of the solvent and the position of the emission spectrum. Moreover, the fluorescence quantum yields were larger in aprotic solvents as compared to protic solvents. This analysis underscores the importance of polarity and hydrogen-bonding environment on the spectroscopic properties of AG-1478. These studies will assume relevance in understanding the interaction of AG-1478 in vitro and in vivo.

  15. Spleen tyrosine kinase contributes to acute renal allograft rejection in the rat.

    Science.gov (United States)

    Ramessur Chandran, Sharmila; Tesch, Greg H; Han, Yingjie; Woodman, Naomi; Mulley, William R; Kanellis, John; Blease, Kate; Ma, Frank Y; Nikolic-Paterson, David J

    2015-02-01

    Kidney allografts induce strong T-cell and antibody responses which mediate acute rejection. Spleen tyrosine kinase (Syk) is expressed by most leucocytes, except mature T cells, and is involved in intracellular signalling following activation of the Fcγ-receptor, B-cell receptor and some integrins. A role for Syk signalling has been established in antibody-dependent native kidney disease, but little is known of Syk in acute renal allograft rejection. Sprague-Dawley rats underwent bilateral nephrectomy and received an orthotopic Wistar renal allograft. Recipient rats were treated with a Syk inhibitor (CC0482417, 30 mg/kg/bid), or vehicle, from 1 h before surgery until being killed 5 days later. Vehicle-treated recipients developed severe allograft failure with marked histologic damage in association with dense leucocyte infiltration (T cells, macrophages, neutrophils and NK cells) and deposition of IgM, IgG and C3. Immunostaining identified Syk expression by many infiltrating leucocytes. CC0482417 treatment significantly improved allograft function and reduced histologic damage, although allograft injury was still clearly evident. CC0482417 failed to prevent T-cell infiltration and activation within the allograft. However, CC0482417 significantly attenuated acute tubular necrosis, infiltration of macrophages and neutrophils and thrombosis of peritubular capillaries. In conclusion, this study identifies a role for Syk in acute renal allograft rejection. Syk inhibition may be a useful addition to T-cell-based immunotherapy in renal transplantation.

  16. [Receptor tyrosine kinase KIT may regulate expression of genes involved in spontaneous regression of neuroblastoma].

    Science.gov (United States)

    Lebedev, T D; Spirin, P V; Suntsova, M V; Ivanova, A V; Buzdin, A A; Prokofjeva, M M; Rubtsov, P M; Prassolov, V S

    2015-01-01

    Hallmark of neuroblastoma is an ability of this malignant tumor to undergo spontaneous regression or differentiation into benign tumor during any stage of the disease, but it is little known about mechanisms of these phenomena. We studied effect of receptor tyrosine kinase receptor KIT on expression of genes, which may be involved in tumor spontaneous regression. Downregulation of KIT expression by RNA interference in SH-SY5Y cells causes suppression of neurotrophin receptor NGFR expression that may promote the loss of sensibility of cells to nerve growth factors, also it causes upregulation of TrkA receptor expression which can stimulate cell differentiation or apoptosis in NGF dependent manner. Furthermore there is an upregulation of genes which stimulate malignant cell detection by immune system, such as genes of major histocompatibility complex HLA class I HLA-B and HLA-C, and interferon-γ receptors IFNGR1 and IFNGR2 genes. Thus KIT can mediate neuroblastoma cell sensibility to neurotrophins and immune system components--two factors directly contributing to spontaneous regression of neuroblastoma.

  17. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor

    Science.gov (United States)

    Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie

    2016-01-01

    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered “off-target” effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo. PMID:27036020

  18. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor.

    Science.gov (United States)

    Lotz-Jenne, Carina; Lüthi, Urs; Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie

    2016-05-03

    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered "off-target" effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo.

  19. [Efficacy of levocarnitine for tyrosine kinase inhibitor-induced painful muscle cramps in patients with chronic myelogenous leukemia].

    Science.gov (United States)

    Yamada, Michiko; Kuroda, Hiroyuki; Shimoyama, Saori; Ito, Ryo; Sugama, Yusuke; Sato, Ken; Yamauchi, Natsumi; Horiguchi, Hiroto; Nakamura, Hajime; Hamaguchi, Kota; Abe, Tomoyuki; Fujii, Shigeyuki; Maeda, Masahiro; Kato, Junji

    2016-04-01

    Muscle cramps are side effects commonly associated with tyrosine kinase inhibitor (TKI) treatment. Patients suffering from muscle cramps are treated with various medications such as calcium, magnesium and vitamin supplements, but these therapies are often ineffective. We report two patients with chronic myelogenous leukemia who developed muscle cramps caused by TKI. These patients were treated successfully with levocarnitine. Both of our cases revealed the beneficial effects of levocarnitine treatment on TKI-induced muscle cramps.

  20. Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

    Directory of Open Access Journals (Sweden)

    Sundler Roger

    2006-05-01

    Full Text Available Abstract Background Yeast and bacteria elicit arachidonate release in macrophages, leading to the formation of leukotrienes and prostaglandins, important mediators of inflammation. Receptors recognising various microbes have been identified, but the signalling pathways are not entirely understood. Cytosolic phospholipase A2 is a major down-stream target and this enzyme is regulated by both phosphorylation and an increase in intracellular Ca2+. Potential signal components are MAP kinases, phosphatidylinositol 3-kinase and phospholipase Cγ2. The latter can undergo tyrosine phosphorylation, and Src family kinases might carry out this phosphorylation. Btk, a Tec family kinase, could also be important. Our aim was to further elucidate the role of Src family kinases and Btk. Methods Arachidonate release from murine peritoneal macrophages was measured by prior radiolabeling. Furthermore, immunoprecipitation and Western blotting were used to monitor changes in activity/phosphorylation of intermediate signal components. To determine the role of Src family kinases two different inhibitors with broad specificity (PP2 and the Src kinase inhibitor 1, SKI-1 were used as well as the Btk inhibitor LFM-A13. Results Arachidonate release initiated by either Staphylococcus aureus or yeast-derived zymosan beads was shown to depend on members of the Src kinase family as well as Btk. Src kinases were found to act upstream of Btk, phosphatidylinositol 3-kinase, phospholipase Cγ2 and the MAP kinases ERK and p38, thereby affecting all branches of the signalling investigated. In contrast, Btk was not involved in the activation of the MAP-kinases. Since the cytosolic phospholipase A2 in macrophages is regulated by both phosphorylation (via ERK and p38 and an increase in intracellular Ca2+, we propose that members of the Src kinase family are involved in both types of regulation, while the role of Btk may be restricted to the latter type. Conclusion Arachidonate release

  1. Protein tyrosine kinases p53/56lyn and p72syk in MHC class I-mediated signal transduction in B lymphoma cells

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Skov, S;

    1998-01-01

    Crosslinking of major histocompatibility complex class I (MHC-I) molecules on the surface of human B lymphoma cells was shown to induce protein tyrosine phosphorylation and mobilization of intracellular free calcium. Immunoprecipitations indicated that the protein tyrosine kinases p53/56lyn and p72......syk are among the tyrosine-phosphorylated proteins. The kinetics of phosphorylation of these kinases after MHC-I crosslinking differ from the kinetics observed after crosslinking of the B cell antigen receptor (BCR). Additional experiments were performed with chicken lyn- and syk-negative DT40 B cells...... and the results indicate that these two kinases have different substrate specificity and regulate intracellular free calcium differently in response to MHC-I crosslinking. In addition MHC-I crosslinking of a sIgM-negative DT40 chicken B cell variant results in less activity of tyrosine kinases and less...

  2. Reduced intensity conditioning is superior to nonmyeloablative conditioning for older chronic myelogenous leukemia patients undergoing hematopoietic cell transplant during the tyrosine kinase inhibitor era

    DEFF Research Database (Denmark)

    Warlick, Erica; Ahn, Kwang Woo; Pedersen, Tanya L;

    2012-01-01

    Tyrosine kinase inhibitors (TKIs) and reduced intensity conditioning (RIC)/nonmyeloablative (NMA) conditioning hematopoietic cell transplants (HCTs) have changed the therapeutic strategy for chronic myelogenous leukemia (CML) patients. We analyzed post-HCT outcomes of 306 CML patients reported to...

  3. DDR2 plays a role in fibroblast migration independent of adhesion ligand and collagen activated DDR2 tyrosine kinase.

    Science.gov (United States)

    Herrera-Herrera, Mireya Liliana; Quezada-Calvillo, Roberto

    2012-12-07

    Discoidin domain receptor-2 (DDR2) is a cell surface tyrosine kinase receptor that can be activated by soluble collagen and has been implicated in diverse physiological functions including organism growth and wound repair. In the current studies, we used fibronectin and collagen-coated 2D surfaces and collagen matrices in combination with siRNA technology to investigate the role of DDR2 in a range of fibroblast motile activities. Silencing DDR2 with siRNA inhibited cell spreading and migration, and similar inhibition occurred regardless whether cells were interacting with fibronectin or collagen surfaces. Under the assay conditions used, DDR2 tyrosine kinase activation was not observed unless soluble collagen was added to the incubation medium. Finally silencing DDR2 also inhibited human fibroblast migration in 3D collagen matrices but had no effect on 3D collagen matrix remodeling and contraction. Taken together, our findings suggest that DDR2 is required for normal fibroblast spreading and migration independent of adhesion ligand and collagen activation of DDR2 tyrosine kinase.

  4. Molecular variation and evolution of the tyrosine kinase domains of insulin receptor IRa and IRb genes in Cyprinidae.

    Science.gov (United States)

    Kong, XiangHui; Wang, XuZhen; He, ShunPing

    2011-07-01

    The insulin receptor (IR) gene plays an important role in regulating cell growth, differentiation and development. In the present study, DNA sequences of insulin receptor genes, IRa and IRb, were amplified and sequenced from 37 representative species of the Cyprinidae and from five outgroup species from non-cyprinid Cypriniformes. Based on coding sequences (CDS) of tyrosine kinase regions of IRa and IRb, molecular evolution and phylogenetic relationships were analyzed to better understand the characteristics of IR gene divergence in the family Cyprinidae. IRa and IRb were clustered into one lineage in the gene tree of the IR gene family, reconstructed using the unweighted pair group method with arithmetic mean (UPGMA). IRa and IRb have evolved into distinct genes after IR gene duplication in Cyprinidae. For each gene, molecular evolution analyses showed that there was no significant difference among different groups in the reconstructed maximum parsimony (MP) tree of Cyprinidae; IRa and IRb have been subjected to similar evolutionary pressure among different lineages. Although the amino acid sequences of IRa and IRb tyrosine kinase regions were highly conserved, our analyses showed that there were clear sequence variations between the tyrosine kinase regions of IRa and IRb proteins. This indicates that IRa and IRb proteins might play different roles in the insulin signaling pathway.

  5. Receptor tyrosine kinase (c-Kit inhibitors: a potential therapeutic target in cancer cells

    Directory of Open Access Journals (Sweden)

    Abbaspour Babaei M

    2016-08-01

    Full Text Available Maryam Abbaspour Babaei,1 Behnam Kamalidehghan,2,3 Mohammad Saleem,4–6 Hasniza Zaman Huri,1,7 Fatemeh Ahmadipour1 1Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 2Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB, Shahrak-e Pajoohesh, 3Medical Genetics Department, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 4Department of Urology, 5Department of Laboratory Medicine and Pathology, Masonic Cancer Center, University of Minnesota, 6Section of Molecular Therapeutics & Cancer Health Disparity, The Hormel Institute, Austin, MN, USA; 7Clinical Investigation Centre, University Malaya Medical Centre, Lembah Pantai, Kuala Lumpur, Malaysia Abstract: c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c

  6. MD-2 interacts with Lyn kinase and is tyrosine phosphorylated following LPS-induced activation of the Toll-like receptor 4 signaling pathway

    Science.gov (United States)

    Gray, Pearl; Dagvadorj, Jargalsaikhan; Michelsen, Kathrin S.; Brikos, Constantinos; Rentsendorj, Altan; Town, Terrence; Crother, Timothy R.; Arditi, Moshe

    2011-01-01

    Stimulation with LPS induces tyrosine phosphorylation of numerous proteins involved in the TLR signaling pathway. In this study, we demonstrate that MD-2 is also tyrosine phosphorylated following LPS stimulation. LPS-induced tyrosine phosphorylation of MD-2 is specific, it is blocked by the tyrosine kinase inhibitor, Herbimycin A, and by an inhibitor of endocytosis, Cytochalsin-D, suggesting that MD-2 phosphorylation occurs during trafficking of MD2 and not on cell surface. Furthermore, we identify two possible phospho-accepting tyrosine residues at positions 22 and 131. Mutant proteins in which these tyrosines were changed to phenylalanine have reduced phosphorylation and significantly diminished ability to activate NF-κB in response to LPS. In addition, MD2 co-precipitates and colocalizes with Lyn kinase, most likely in ER. A Lyn-binding peptide inhibitor abolished MD2 tyrosine phosphorylation, suggesting that Lyn is a likely candidate to be the kinase required for MD-2 tyrosine phophorylation. Our study demonstrates that tyrosine phosphorylation of MD-2 is important for signaling following exposure to LPS and underscores the importance of this event in mediating an efficient and prompt immune response. PMID:21918188

  7. Myeloid differentiation factor-2 interacts with Lyn kinase and is tyrosine phosphorylated following lipopolysaccharide-induced activation of the TLR4 signaling pathway.

    Science.gov (United States)

    Gray, Pearl; Dagvadorj, Jargalsaikhan; Michelsen, Kathrin S; Brikos, Constantinos; Rentsendorj, Altan; Town, Terrence; Crother, Timothy R; Arditi, Moshe

    2011-10-15

    Stimulation with LPS induces tyrosine phosphorylation of numerous proteins involved in the TLR signaling pathway. In this study, we demonstrated that myeloid differentiation factor-2 (MD-2) is also tyrosine phosphorylated following LPS stimulation. LPS-induced tyrosine phosphorylation of MD-2 is specific; it is blocked by the tyrosine kinase inhibitor, herbimycin A, as well as by an inhibitor of endocytosis, cytochalasin D, suggesting that MD-2 phosphorylation occurs during trafficking of MD-2 and not on the cell surface. Furthermore, we identified two possible phospho-accepting tyrosine residues at positions 22 and 131. Mutant proteins in which these tyrosines were changed to phenylalanine had reduced phosphorylation and significantly diminished ability to activate NF-κB in response to LPS. In addition, MD-2 coprecipitated and colocalized with Lyn kinase, most likely in the endoplasmic reticulum. A Lyn-binding peptide inhibitor abolished MD-2 tyrosine phosphorylation, suggesting that Lyn is a likely candidate to be the kinase required for MD-2 tyrosine phosphorylation. Our study demonstrated that tyrosine phosphorylation of MD-2 is important for signaling following exposure to LPS and underscores the importance of this event in mediating an efficient and prompt immune response.

  8. Carcinoma of an unknown primary: are EGF receptor, Her-2/neu, and c-Kit tyrosine kinases potential targets for therapy?

    OpenAIRE

    Massard, C; Voigt, J-J; Laplanche, A; Culine, S; Lortholary, A; Bugat, R; Theodore, C.; Priou, F; Kaminsky, M-C; Lesimple, T; Pivot, X; B. Coudert; Douillard, J-Y; Merrouche, Y; Fizazi, K

    2007-01-01

    Carcinomas of an unknown primary site (CUP) are heterogeneous tumours with a median survival of only 8 months. Tyrosine kinase inhibitors are promising new drugs. The aim of this study was to determine the expression of EGF-receptor, Her-2/neu, and c-Kit tyrosine kinases in CUP. Paraffin-embedded specimens were obtained from 54 patients with a CUP who were included in the GEFCAPI 01 randomised phase II trial. Immunohistochemistry was performed using the Dako autostainer with antibodies direct...

  9. The expression of COX-2 in VEGF-treated endothelial cells is mediated through protein tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Pravit Akarasereenont

    2002-01-01

    Full Text Available Cyclooxygenase (COX, existing as the COX-1 and COX-2 isoforms, converts arachidonic acid to prostaglandin H2, which is then further metabolized to various prostaglandins. Vascular endothelial growth factor (VEGF has been shown to play important roles in inflammation and is upregulated by the prostaglandin E series through COX-2 in several cell types. Here, we have investigated the effects of VEGF on the COX isoform expressed in human umbilical vein endothelial cells (HUVEC. The signalling mechanism of the COX isoform expressed in endothelial cells activated with VEGF will be also investigated using the tyrosine kinase inhibitor, genistein, and protein kinase C inhibitor, staurosporine. The activity of COX2 was assessed by measuring the production of 6-keto-prostaglandin F1α in the presence of exogenous arachidonic acids (10 μM, 10 min by enzyme immunoassay. The expression of COX isoform protein was detected by immunoblot using specific antibodies. Untreated HUVEC contained no COX-2 protein. In HUVEC treated with VEGF (0.01-50 ng/ml, COX-2 protein, but not COX-1, and COX activity were increased in a dose-dependent manner. Interestingly, the increased COX-2 protein and activity in response to VEGF (10 ng/ml was inhibited by the tyrosine kinase inhibitor, genistein (0.05-5 μg/ml, but not by the protein kinase C inhibitor, staurosporine (0.1-10 ng/ml. Thus, the induction of COX-2 by VEGF in endothelial cells was mediated through protein tyrosine kinase, and the uses of specific COX-2 inhibitors in these conditions, in which VEGF was involved, might have a role.

  10. Interaction of the growth hormone receptor cytoplasmic domain with the JAK2 tyrosine kinase.

    Science.gov (United States)

    Frank, S J; Gilliland, G; Kraft, A S; Arnold, C S

    1994-11-01

    An early step in GH action involves tyrosine phosphorylation of various cellular proteins. Recently, it has been shown in murine preadipocytes that GH promotes the association of its receptor (the GHR) with and the activation of the JAK2 tyrosine kinase. In this study, we confirmed the human (h) GH-induced association of JAK2 with hGHR in IM-9 cells by coimmunoprecipitation experiments using anti-hGHR serum. We further examined the interaction of JAK2 with the GHR cytoplasmic domain by two lines of investigation. For in vitro studies, we assayed by immunoblotting the ability of cell-derived JAK2 to interact with glutathione S-transferase fusion proteins containing elements of the hGHR cytoplasmic domain. A fusion protein containing the entire hGHR cytoplasmic domain (residues 271-620) specifically associated with JAK2 independent of prior stimulation of cells with hGH. This interaction was not dependent on tyrosine phosphorylation of either partner. Mutational analysis of the hGHR cytoplasmic domain component of the fusions indicated that a membrane-proximal 20-residue region that includes the proline-rich box 1 was necessary for the interaction. This region appeared to cooperate with another region(s), largely in the N-terminal one third of the cytoplasmic domain, to promote full interaction with JAK2. For in vivo reconstitution experiments, wild-type (WT) and mutant rabbit GHRs (rGHRs) along with murine JAK2 were expressed by transient transfection in COS-7 cells. rGHR mutations were confined to the cytoplasmic domain and included C-terminal truncations as well as internal deletions of residues 297-406 and 278-292 (the latter contains box 1). All mutant rGHRs were expressed at the cell surface and bound hGH to a degree similar to the WT rGHR. Receptors were tested for their ability to mediate the hGH-induced immunoprecipitability of JAK2 with phosphotyrosine (APT) antibodies. A rGHR truncated to residue 275 [rGHR-(1-275)], which contains only five cytoplasmic

  11. The Bmx tyrosine kinase is activated by IL-3 and G-CSF in a PI-3K dependent manner.

    Science.gov (United States)

    Ekman, N; Arighi, E; Rajantie, I; Saharinen, P; Ristimäki, A; Silvennoinen, O; Alitalo, K

    2000-08-24

    Cytoplasmic protein tyrosine kinases play crucial roles in signaling via a variety of cell surface receptors. The Bmx tyrosine kinase, a member of the Tec family, is expressed in hematopoietic cells of the granulocytic and monocytic lineages. Here we show that Bmx is catalytically activated by interleukin-3 (IL-3) and granulocyte-colony stimulating factor (G-CSF) receptors. Activation of Bmx required phosphatidylinositol 3-kinase (PI-3K) as demonstrated by the ability of PI-3K inhibitors to block the activation signal. A green fluorescent protein (GFP) tagged Bmx was translocated to cellular membranes upon co-expression of a constitutively active form of PI-3K, further indicating a role for PI-3K in signaling upstream of Bmx. The expression of wild type Bmx in 32D myeloid progenitor cells resulted in apoptosis in the presence of G-CSF, while cells expressing a kinase dead mutant of Bmx differentiated into mature granulocytes. However, Bmx did not modulate IL-3-dependent proliferation of the cells. These results demonstrate distinct effects of Bmx in cytokine induced proliferation and differentiation of myeloid cells, and suggest that the stage specific expression of Bmx is critical for the differentiation of myeloid cells. Oncogene (2000) 19, 4151 - 4158

  12. Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hyeon-Ok [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Hong, Sung-Eun [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Kim, Chang Soon [Department of Microbiological Engineering, Kon-Kuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 143–701 (Korea, Republic of); Park, Jin-Ah; Kim, Jin-Hee; Kim, Ji-Young; Kim, Bora [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Chang, Yoon Hwan; Hong, Seok-Il; Hong, Young Jun [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Park, In-Chul, E-mail: parkic@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Lee, Jin Kyung, E-mail: jklee@kirams.re.kr [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of)

    2015-08-15

    Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H + ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. - Highlights: • Co-treatment with EGFR TKIs and vATPase inhibitors induces synergistic cell death • EGFR TKIs enhance cell sensitivity to vATPase inhibitors via Hif-1α downregulation • Co-treatment of these inhibitors is potentially effective for the treatment of NSCLC.

  13. The expression andprognostic value ofprotein tyrosine kinase 6 inearly-stage cervical squamous cell cancer

    Institute of Scientific and Technical Information of China (English)

    XiaoJingWang; YingXiong; ZeBiaoMa; JianChuanXia; YanFangLi

    2016-01-01

    Background:Protein tyrosine kinase 6 (PTK6) is overexpressed in many epithelial tumors and predicts poor progno‑sis. However, PTK6 expression status and its role in cervical squamous cell cancer are unknown. This study aimed to investigate the expression level and clinical signiifcance of PTK6 in early‑stage cervical squamous cell cancer. Methods:Quantitative reverse transcription‑polymerase chain reaction (qRT‑PCR) and western blotting analysis were performed to detect PTK6 mRNA and protein expression levels in 10 freshly frozen, early‑stage cervical squamous cell cancer specimens and adjacent non‑tumorous cervical tissues. The expression of PTK6 was detected using immuno‑histochemical staining in 150 formalin‑ifxed, paraffn‑embedded, early‑stage cervical squamous cell cancer sections and 10 normal cervical tissue sections. Results:The mRNA and protein levels of PTK6 in cancer tissues were higher than those in adjacent non‑tumorous cervical tissues. Immunohistochemical analysis showed that PTK6 was not expressed in normal cervical tissues but was overexpressed in the cytoplasm of cervical squamous cell cancer cells. The level of PTK6 expression was signiif‑cantly associated with tumor grade (P=0.020). The 5‑year overall survival rate of patients with high PTK6 expression was lower than that of patients with low PTK6 expression (81.3% vs. 96.2%,P=0.008). Multivariate Cox regression analysis showed that the expression level of PTK6 in cervical squamous cell cancer was an independent prognostic factor for patient survival (hazard ratio=5.999, 95% conifdence interval 1.622–22.191,P Conclusions:PTK6 is overexpressed in cervical squamous cell cancer. Increased PTK6 expression is associated with reduced 5‑year overall survival. PTK6 expression is an independent prognostic predictor for cervical cancer.

  14. The tyrosine kinase inhibitor PD153035: implication of labeling position on radiometabolites formed in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Samen, Erik [Karolinska Pharmacy, Karolinska University Hospital Solna, SE-17176 Stockholm (Sweden); Thorell, Jan-Olov [Karolinska Pharmacy, Karolinska University Hospital Solna, SE-17176 Stockholm (Sweden); Fredriksson, Anna [Karolinska Pharmacy, Karolinska University Hospital Solna, SE-17176 Stockholm (Sweden); Stone-Elander, Sharon [Karolinska Pharmacy, Karolinska University Hospital Solna, SE-17176 Stockholm (Sweden) and Department Clinical Neurosciences, Karolinska Institutet, Karolinska University Hospital Solna, SE-17176 Stockholm (Sweden)]. E-mail: sharon.stone-elander@karolinska.se

    2006-11-15

    Introduction: The epidermal growth factor receptor is highly expressed in several types of cancers. Molecules with high affinity to its intracellular tyrosine kinase domain are being developed as in vivo imaging probes. The 4-anilinoquinazoline PD153035 has promising in vitro and in vivo properties for development as a reversible radioligand. Labeling it with carbon-11 in either of its two methoxy positions can potentially give rise to different radiometabolites and, consequently, different imaging capabilities. An evaluation of the radiotracers' metabolism was needed to determine the potential significance of the labeling position. Methods: PD153035 was labeled in the 6- and 7-O-methoxy positions by reacting the corresponding O-desmethyl precursors with [{sup 11}C]methyl iodide. The two radiolabeled compounds were each incubated for 1 h with human and rat liver microsomes. At five time points, the radiolabeled metabolites were examined using radio-liquid chromatography. One metabolite was isolated and subjected to mass spectroscopic analysis. Results: A major polar metabolite was obtained in all incubations. Its molecular weight was consistent with an addition of oxygen, and its fragmentation was consistent with an N-oxidation rather than an aromatic hydroxylation. Regioselective 7-O-dealkylation was also observed, albeit in substantial amounts only in the assay using human microsomes. Conclusions: Radiolabeling in the 7-O-methoxy position is advocated, since the labeled metabolites produced in the 7-O-demethylation are polar and probably rapidly cleared. The differences observed in the incubations with rat and human microsomes suggest that in vivo positron emission tomography studies with {sup 11}C-labeled PD153035 in rodents may not be directly predictive for studies in humans.

  15. Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia.

    Directory of Open Access Journals (Sweden)

    Mohammad Hojjat-Farsangi

    Full Text Available ROR1 is a receptor tyrosine kinase expressed in chronic lymphocytic leukemia (CLL and several other malignancies but absent in most adult normal tissues. ROR1 is considered an onco-fetal antigen. In the present study we analysed spontaneous humoral and cellular immunity against ROR1 in CLL patients.Antibodies against ROR1 were analysed in 23 patients and 20 healthy donors by ELISA and Western blot. Purified serum IgG from patients was tested for cytotoxicity against CLL cells using the MTT viability assay. A cellular immune response against ROR1 derived HLA-A2 restricted 9 aa and 16 aa long peptides were analysed using peptide loaded dendritic cells co-cultured with autologous T cells from CLL patients (n = 9 and healthy donors (n = 6. IFN-γ, IL-5 and IL-17A-secreting T cells were assessed by ELISPOT and a proliferative response using a H3-thymidine incorporation assay.The majority of CLL patients had antibodies against ROR1. Significantly higher titers of anti-ROR1 antibodies were noted in patients with non-progressive as compared to progressive disease. The extracellular membrane-close ROR1 KNG domain seemed to be an immunodominant epitope. Ten patients with high titers of anti-ROR1 binding antibodies were tested for cytotoxicity. Five of those had cytotoxic anti-ROR1 antibodies against CLL cells. ROR1-specific IFN-γ and IL-17A producing T cells could be detected in CLL patients, preferentially in non-progressive as compared to patients with progressive disease (p<0.05.ROR1 seemed to spontaneously induce a humoral as well as a T cell response in CLL patients. The data support the notion that ROR1 might be a specific neo-antigen and may serve as a target for immunotherapy.

  16. Removal of Soluble Fms-Like Tyrosine Kinase-1 by Dextran Sulfate Apheresis in Preeclampsia.

    Science.gov (United States)

    Thadhani, Ravi; Hagmann, Henning; Schaarschmidt, Wiebke; Roth, Bernhard; Cingoez, Tuelay; Karumanchi, S Ananth; Wenger, Julia; Lucchesi, Kathryn J; Tamez, Hector; Lindner, Tom; Fridman, Alexander; Thome, Ulrich; Kribs, Angela; Danner, Marco; Hamacher, Stefanie; Mallmann, Peter; Stepan, Holger; Benzing, Thomas

    2016-03-01

    Preeclampsia is a devastating complication of pregnancy. Soluble Fms-like tyrosine kinase-1 (sFlt-1) is an antiangiogenic protein believed to mediate the signs and symptoms of preeclampsia. We conducted an open pilot study to evaluate the safety and potential efficacy of therapeutic apheresis with a plasma-specific dextran sulfate column to remove circulating sFlt-1 in 11 pregnant women (20-38 years of age) with very preterm preeclampsia (23-32 weeks of gestation, systolic BP ≥140 mmHg or diastolic BP ≥90 mmHg, new onset protein/creatinine ratio >0.30 g/g, and sFlt-1/placental growth factor ratio >85). We evaluated the extent of sFlt-1 removal, proteinuria reduction, pregnancy continuation, and neonatal and fetal safety of apheresis after one (n=6), two (n=4), or three (n=1) apheresis treatments. Mean sFlt-1 levels were reduced by 18% (range 7%-28%) with concomitant reductions of 44% in protein/creatinine ratios. Pregnancy continued for 8 days (range 2-11) and 15 days (range 11-21) in women treated once and multiple times, respectively, compared with 3 days (range 0-14) in untreated contemporaneous preeclampsia controls (n=22). Transient maternal BP reduction during apheresis was managed by withholding pre-apheresis antihypertensive therapy, saline prehydration, and reducing blood flow through the apheresis column. Compared with infants born prematurely to untreated women with and without preeclampsia (n=22 per group), no adverse effects of apheresis were observed. In conclusion, therapeutic apheresis reduced circulating sFlt-1 and proteinuria in women with very preterm preeclampsia and appeared to prolong pregnancy without major adverse maternal or fetal consequences. A controlled trial is warranted to confirm these findings.

  17. Intestinal colonization by Candida albicans alters inflammatory responses in Bruton's tyrosine kinase-deficient mice.

    Directory of Open Access Journals (Sweden)

    Karin Strijbis

    Full Text Available The commensal yeast Candida albicans is part of the human intestinal microflora and is considered a "pathobiont", a resident microbe with pathogenic potential yet harmless under normal conditions. The aim of this study was to investigate the effect of C. albicans on inflammation of the intestinal tract and the role of Bruton's tyrosine kinase (Btk. Btk is an enzyme that modulates downstream signaling of multiple receptors involved in innate and adaptive immunity, including the major anti-fungal receptor Dectin-1. Colitis was induced in wild type and Btk-/- mice by treatment with dextran sodium sulfate (DSS and the gastrointestinal tract of selected treatment groups were then colonized with C. albicans. Colonization by C. albicans neither dampened nor exacerbated inflammation in wild type mice, but colon length and spleen weight were improved in Btk-deficient mice colonized with C. albicans. Neutrophil infiltration was comparable between wild type and Btk-/- mice, but the knockout mice displayed severely reduced numbers of macrophages in the colon during both DSS and DSS/Candida treatment. Smaller numbers and reduced responsiveness of Btk-/- macrophages might partially explain the improved colon length of Btk-/- mice as a result of Candida colonization. Surprisingly, DSS/Candida-treated Btk-/- animals had higher levels of certain pro-inflammatory cytokines and levels of the anti-inflammatory cytokine TGF-β were reduced compared to wild type. A clustering and correlation analysis showed that for wild type animals, spleen TGF-β and colon IL-10 and for Btk-/- spleen and colon levels of IL-17A best correlated with the inflammatory parameters. We conclude that in Btk-/- immunocompromised animals, colonization of the gastrointestinal tract by the commensal yeast C. albicans alters inflammatory symptoms associated with colitis.

  18. Role of ErbB family receptor tyrosine kinases in intrahepatic cholangiocarcinoma

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Aberrant expression and signaling of epidermal growth factor receptor (ErbB) family receptor tyrosine kinases, most notably that of ErbB2 and ErbB1, have been implicated in the molecular pathogenesis of intrahepatic cholangiocarcinoma. Constitutive overexpression of ErbB2 and/or ErbB1 in malignant cholangiocytes has raised interest in the possibility that agents which selectively target these receptors could potentially be effective in cholangiocarcinoma therapy. However, current experience with such ErbB-directed therapies have at best produced only modest responses in patients with biliary tract cancers. This review provides a comprehensive and critical analysis of both preclinical and clinical studies aimed at assessing the role of altered ErbB2 and/or ErbB1 expression, genetic modifications, and dysregulated signaling on cholangiocarcinoma development and progression. Specific limitations in experimental approaches that have been used to assess human cholangiocarcinoma specimens for ErbB2 and/or ErbB1 overexpression and gene amplification are discussed. In addition, current rodent models of intrahepatic cholangiocarcinogenesis associated with constitutive ErbB2 overexpression are reviewed. Select interactive relationships between ErbB2 or ErbB1 with other relevant molecular signaling pathways associated with intrahepatic cholangiocarcinoma development and progression are also detailed, including those linking ErbB receptors to bile acid, cyclooxygenase-2, interleukin-6/gp130, transmembrane mucins, hepatocyte growth factor/Met, and vascular endothelial growth factor signaling. Lastly, various factors that can limit therapeutic efficacy of ErbB-targeted agents against cholangiocarcinoma are considered.

  19. Ror family receptor tyrosine kinases regulate the maintenance of neural progenitor cells in the developing neocortex.

    Science.gov (United States)

    Endo, Mitsuharu; Doi, Ryosuke; Nishita, Michiru; Minami, Yasuhiro

    2012-04-15

    The Ror family receptor tyrosine kinases (RTKs), Ror1 and Ror2, have been shown to play crucial roles in developmental morphogenesis by acting as receptors or co-receptors to mediate Wnt5a-induced signaling. Although Ror1, Ror2 and Wnt5a are expressed in the developing brain, little is known about their roles in the neural development. Here we show that Ror1, Ror2 and their ligand Wnt5a are highly expressed in neocortical neural progenitor cells (NPCs). Small interfering RNA (siRNA)-mediated suppression of Ror1, Ror2 or Wnt5a in cultured NPCs isolated from embryonic neocortex results in the reduction of βIII-tubulin-positive neurons that are produced from NPCs possibly through the generation of T-box brain 2 (Tbr2)-positive intermediate progenitors. BrdU-labeling experiments further reveal that the proportion of proliferative and neurogenic NPCs, which are positive for neural progenitor cell marker (Pax6) but negative for glial cell marker (glial fibrillary acidic protein; GFAP), is reduced within a few days in culture following knockdown of these molecules, suggesting that Ror1, Ror2 and Wnt5a regulate neurogenesis through the maintenance of NPCs. Moreover, we show that Dishevelled 2 (Dvl2) is involved in Wnt5a-Ror1 and Wnt5a-Ror2 signaling in NPCs, and that suppressed expression of Dvl2 indeed reduces the proportion of proliferative and neurogenic NPCs. Interestingly, suppressed expression of either Ror1 or Ror2 in NPCs in the developing neocortex results in the precocious differentiation of NPCs into neurons, and their forced expression results in delayed differentiation. Collectively, these results indicate that Wnt5a-Ror1 and Wnt5a-Ror2 signaling pathways play roles in maintaining proliferative and neurogenic NPCs during neurogenesis of the developing neocortex.

  20. Alignment independent 3D-QSAR, quantum calculations and molecular docking of Mer specific tyrosine kinase inhibitors as anticancer drugs

    Directory of Open Access Journals (Sweden)

    Fereshteh Shiri

    2016-03-01

    Full Text Available Mer receptor tyrosine kinase is a promising novel cancer therapeutic target in many human cancers, because abnormal activation of Mer has been implicated in survival signaling and chemoresistance. 3D-QSAR analyses based on alignment independent descriptors were performed on a series of 81 Mer specific tyrosine kinase inhibitors. The fractional factorial design (FFD and the enhanced replacement method (ERM were applied and tested as variable selection algorithms for the selection of optimal subsets of molecular descriptors from a much greater pool of such regression variables. The data set was split into 65 molecules as the training set and 16 compounds as the test set. All descriptors were generated by using the GRid INdependent descriptors (GRIND approach. After variable selection, GRIND were correlated with activity values (pIC50 by PLS regression. Of the two applied variable selection methods, ERM had a noticeable improvement on the statistical parameters of PLS model, and yielded a q2 value of 0.77, an rpred2 of 0.94, and a low RMSEP value of 0.25. The GRIND information contents influencing the affinity on Mer specific tyrosine kinase were also confirmed by docking studies. In a quantum calculation study, the energy difference between HOMO and LUMO (gap implied the high interaction of the most active molecule in the active site of the protein. In addition, the molecular electrostatic potential energy at DFT level confirmed results obtained from the molecular docking. The identified key features obtained from the molecular modeling, enabled us to design novel kinase inhibitors.

  1. Rescue of a trafficking defective human pacemaker channel via a novel mechanism: roles of Src, Fyn, and Yes tyrosine kinases.

    Science.gov (United States)

    Lin, Yen-Chang; Huang, Jianying; Kan, Hong; Frisbee, Jefferson C; Yu, Han-Gang

    2009-10-30

    Therapeutic strategies such as using channel blockers and reducing culture temperature have been used to rescue some long QT-associated voltage-gated potassium Kv trafficking defective mutant channels. A hyperpolarization-activated cyclic nucleotide-gated HCN4 pacemaker channel mutant (D553N) has been recently found in a patient associated with cardiac arrhythmias including long QT. D553N showed the defective trafficking to the cell surface, leading to little ionic current expression (loss-of-function). We show in this report that enhanced tyrosine phosphorylation mediated by Src, Fyn, and Yes kinases was able to restore the surface expression of D553N for normal current expression. Src or Yes, but not Fyn, significantly increased the current density and surface expression of D553N. Fyn accelerated the activation kinetics of the rescued D553N. Co-expression of D553N with Yes exhibited the slowest activation kinetics of D553N. Src, Fyn, and Yes significantly enhanced the tyrosine phosphorylation of D553N. A combination of Src, Fyn, and Yes rescued the current expression and the gating of D553N comparable with those of wild-type HCN4. In conclusion, we demonstrate a novel mechanism using three endogenous Src kinases to rescue a trafficking defective HCN4 mutant channel (D553N) by enhancing the tyrosine phosphorylation of the mutant channel protein.

  2. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    Directory of Open Access Journals (Sweden)

    Meehan Maria

    2012-02-01

    Full Text Available Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA, an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA's primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  3. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    LENUS (Irish Health Repository)

    Meehan, Maria

    2012-02-05

    Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA\\'s primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  4. Growth-Factor-Driven Rescue to Receptor Tyrosine Kinase (RTK) Inhibitors through Akt and Erk Phosphorylation in Pediatric Low Grade Astrocytoma and Ependymoma

    NARCIS (Netherlands)

    Sie, Mariska; den Dunnen, Wilfred F. A.; Lourens, Harm Jan; Meeuwsen-de Boer, Tiny G. J.; Scherpen, Frank J. G.; Zomerman, Walderik W.; Kampen, Kim R.; Hoving, Eelco W.; de Bont, Eveline S. J. M.

    2015-01-01

    Up to now, several clinical studies have been started investigating the relevance of receptor tyrosine kinase (RTK) inhibitors upon progression free survival in various pediatric brain tumors. However, single targeted kinase inhibition failed, possibly due to tumor resistance mechanisms. The present

  5. The tyrosine kinase receptor c-met, its cognate ligand HGF and the tyrosine kinase receptor trasducers STAT3, PI3K and RHO in thyroid nodules associated with Hashimoto's thyroiditis: an immunohistochemical characterization

    Directory of Open Access Journals (Sweden)

    R. M. Ruggeri

    2010-06-01

    Full Text Available Hepatocyte growth factor (HGF exerts proliferative activities in thyrocytes upon binding to its tyrosine kinase receptor c-met and is also expressed in benign thyroid nodules as well as in Hashimoto’s thyroiditis (HT. The simultaneous expression of HGF/c-met and three trasducers of tyrosine kinase receptors (STAT3, PI3K, RHO in both the nodular and extranodular tissues were studied by immunohistochemistry in 50 benign thyroid nodules (NGs, 25 of which associated with HT. The ligand/tyrosine kinase receptor pair HGF/c-met and the two trasducers PI3K and RHO were expressed in NGs, regardless of association with HT, with a higher positive cases percentage in HT-associated NGs compared to not HT-associated NGs (25/25 or 100% vs 7/25 or 28%; P<0.001. HGF, PI3K and RHO expression was only stromal (fibroblasts and endothelial cells, in all 32 reactive NGs, while c-met localization was consistently epithelial (thyrocyes. Immu­noreactions for HGF, c-met, PI3K and RHO were also apparent in the extra-nodular tissue of HT specimens, where HGF and PI3K were expressed not only in stromal cells but also in thyrocyes along with the c-met. Finally, a positive correlation was observed between the proportion of HGF, c-met, PI3K follicular cells and the grade of lymphoid aggregates in HT. In conclusion, HGF, c-met, PI3K are much more frequently and highly expressed in HT compared to NGs, and among all NGs in those present in the context of HT. A paracrine effect of HFG/c-met on nodule development, based on the prevalent stromal expression, may be suggested along with a major role of HGF/c-met and PI3K in HT. Finally, the expression of such molecules in HT may be regulated by lymphoid infiltrate.

  6. NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells.

    Science.gov (United States)

    Ambrogio, Chiara; Martinengo, Cinzia; Voena, Claudia; Tondat, Fabrizio; Riera, Ludovica; di Celle, Paola Francia; Inghirami, Giorgio; Chiarle, Roberto

    2009-11-15

    Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell-specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell-specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor-related signaling molecules, including CD3epsilon, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALK(K210R), downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling.

  7. Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src.

    Directory of Open Access Journals (Sweden)

    Silviya R Stateva

    Full Text Available Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W-7 inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR, in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.

  8. Glycine-extended gastrin activates two independent tyrosine-kinases in upstream of p85/p110 phosphatidylinositol 3-kinase in human colonic tumour cells

    Institute of Scientific and Technical Information of China (English)

    Audrey Ferrand; Aline Kowalski-Chauvel; Julie Pannequin; Claudine Bertrand; Daniel Fourmy; Marlene Dufresne; Catherine Seva

    2006-01-01

    AIM: To investigate whether Src, JAK2 and phosphatidylinositol 3-kinase (PI3K) pathways are involved in the proliferation of human colonic tumour cells induced by glycine-extended gastrin (G-gly), the precursor of the mature amidated gastrin and to elucidate the molecular interaction between these three kinases in response to this peptide.METHODS: Using the human colonic tumour cell line HCT116 as a model, we first measured the activation of PI3K, p60-Src and JAK2 in response to G-gly by in vitro kinase assays. Then we investigated the involvement of these kinases in G-gly-induced cell proliferation by MTT test.RESULTS: G-gly stimulation induced p60-Src, JAK2 and PI3K activation in HCT116. The different pathways were involved in proliferation of human colon cancer cells induced by G-gly. Furthermore, we found that both Src and JAK2 were necessary to PI3K regulation by this peptide. However, we did not find any cross-talk between the two tyrosine kinases.CONCLUSION: Our results suggest that the p60-Src/ PI3K and JAK2/PI3K pathways act independently to mediate G-gly proliferative effect on human colonic tumour cells.

  9. HER2 Oncogenic Function Escapes EGFR Tyrosine Kinase Inhibitors via Activation of Alternative HER Receptors in Breast Cancer Cells

    Science.gov (United States)

    Kong, Anthony; Calleja, Véronique; Leboucher, Pierre; Harris, Adrian; Parker, Peter J.; Larijani, Banafshé

    2008-01-01

    Background The response rate to EGFR tyrosine kinase inhibitors (TKIs) may be poor and unpredictable in cancer patients with EGFR expression itself being an inadequate response indicator. There is limited understanding of the mechanisms underlying this resistance. Furthermore, although TKIs suppress the growth of HER2-overexpressing breast tumor cells, they do not fully inhibit HER2 oncogenic function at physiological doses. Methodology and Principal Findings Here we have provided a molecular mechanism of how HER2 oncogenic function escapes TKIs' inhibition via alternative HER receptor activation as a result of autocrine ligand release. Using both Förster Resonance Energy Transfer (FRET) which monitors in situ HER receptor phosphorylation as well as classical biochemical analysis, we have shown that the specific tyrosine kinase inhibitors (TKIs) of EGFR, AG1478 and Iressa (Gefitinib) decreased EGFR and HER3 phosphorylation through the inhibition of EGFR/HER3 dimerization. Consequent to this, we demonstrate that cleavage of HER4 and dimerization of HER4/HER2 occur together with reactivation of HER3 via HER2/HER3, leading to persistent HER2 phosphorylation in the now resistant, surviving cells. These drug treatment–induced processes were found to be mediated by the release of ligands including heregulin and betacellulin that activate HER3 and HER4 via HER2. Whereas an anti-betacellulin antibody in combination with Iressa increased the anti-proliferative effect in resistant cells, ligands such as heregulin and betacellulin rendered sensitive SKBR3 cells resistant to Iressa. Conclusions and Significance These results demonstrate the role of drug-induced autocrine events leading to the activation of alternative HER receptors in maintaining HER2 phosphorylation and in mediating resistance to EGFR tyrosine kinase inhibitors (TKIs) in breast cancer cells, and hence specify treatment opportunities to overcome resistance in patients. PMID:18682844

  10. Alteration and potential role of soluble fms-like tyrosine kinase receptor 1 in preeclampsia

    Institute of Scientific and Technical Information of China (English)

    LI Yi; LI Dong-hong; YAO Yuan-qing; ZHAO Hong-xi; ZHANG Chun-li; HUANG Liang

    2005-01-01

    Objective:To investigate the alteration of serum soluble fms-like tyrosine kinase receptor 1 (sFlt-1), the possible cause in preeclampsia, the relationship between altered sFlt-1 and the development of pregnancy. Methods: Semi-quantitative RT-PCR was carried out to detect the level of sFlt-1 mRNA in placental tissue of 10 preeclampsia (study group) and 10 normotensive pregnancies (control group); ELISA was used to detect the serum level of sFlt-1 in peripheral venous blood and uterine venous blood in control group (n=35) and study group (n=35), and furthermore to detect the volume of peripheral venous blood sFlt-1 in early, middle and later pregnancies. Results: sFlt-1 mRNA of placental tissue was significantly higher in preeclampsia than that in normal pregnancy. The serum level of sFlt-1 of peripheral vein in preeclampsia was higher than that in normal pregnancy ([ 5639.6±3190.5] pg/ml vs[ 2194.4±635.8 ] pg/ml, P<0.01) , so did the serum sFlt-1 of uterine vein in preeclampsia and control group ([7673.3±2295.8] pg/ml vs[ 3056.6±784.6] pg/ml, P<0.01), indicating that the volume of sFlt-1 of uterine vein blood was significantly higher than that of peripheral venous blood (P<0.01). The serum level of sFlt-1 in early, middle and later pregnancy were (31.7±19.6) pg/ml (n=10), (994.0±302.1) pg/ml (n=10), (2194.4±635.8) pg/ml (n=35), respectively, showing that the level of sFlt-1 in peripheral venous blood increasingly elevated with the development of pregnancy (P<0.01). Conclusion: Preeclampsia is associated with increased level of sFlt-1 which might be largely from placenta during pregnancy.

  11. Tyrosine kinase domain mutations of EGFR gene in head and neck squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Vatte C

    2017-03-01

    Full Text Available Chittibabu Vatte,1 Ali M Al Amri,2 Cyril Cyrus,1 Shahanas Chathoth,1 Sadananda Acharya,3 Tariq Mohammad Hashim,4 Zhara Al Ali,2 Saleh Tawfeeq Alshreadah,2 Ahmed Alsayyah,4 Amein K Al-Ali5 1Department of Genetic Research, Institute for Research and Medical Consultation, University of Dammam, Dammam, 2Department of Internal Medicine, King Fahd Hospital of the University, University of Dammam, Al-Khobar, 3Department of Stemcell Research, Institute for Research and Medical Consultation, 4Department of Pathology, King Fahd Hospital of the University, University of Dammam, Al-Khobar, 5Department of Biochemistry, College of Medicine, University of Dammam, Dammam, Kingdom of Saudi Arabia Background: Epidermal growth factor receptor (EGFR is a commonly altered gene that is identified in various cancers, including head and neck squamous cell carcinoma (HNSCC. Therefore, EGFR is a promising molecular marker targeted by monoclonal antibodies and small molecule inhibitors targeting the tyrosine kinase (TK domain. Objective: The objective of this study was to investigate the spectrum of mutations in exons 18, 19, 20, and 21 of the EGFR gene in HNSCC patients. Materials and methods: This retrospective study included 47 confirmed HNSCC cases. Mutations in the TK domain, exons 18, 19, 20, and 21 of the EGFR gene, were detected by Scorpion® chemistry and ARMS® technologies on Rotor-Gene Q real-time polymerase chain reaction.Results: The tumors exhibited EGFR-TK domain mutations in 57% of cases. Four cases of T790M mutations were reported for the first time among HNSCC patients. Out of the total mutations, L861Q (exon 21, exon 20 insertions and deletions of exon 19 accounted for the majority of mutations (21%, 19%, and 17%, respectively. EGFR mutation status was correlated with the higher grade (P=0.026 and advanced stage (P=0.034 of HNSCC tumors.Conclusion: Higher frequency of EGFR-TK domain mutations together with the presence of the T790M mutation suggests

  12. N-linked glycosylation supports cross-talk between receptor tyrosine kinases and androgen receptor.

    Directory of Open Access Journals (Sweden)

    Harri M Itkonen

    Full Text Available Prostate cancer is the second most common cause of cancer-associated deaths in men and signalling via a transcription factor called androgen receptor (AR is an important driver of the disease. Androgen treatment is known to affect the expression and activity of other oncogenes including receptor tyrosine kinases (RTKs. In this study we report that AR-positive prostate cancer cell-lines express 50% higher levels of enzymes in the hexosamine biosynthesis pathway (HBP than AR-negative prostate cell-lines. HBP produces hexosamines that are used by endoplasmic reticulum and golgi enzymes to glycosylate proteins targeted to plasma-membrane and secretion. Inhibition of O-linked glycosylation by ST045849 or N-linked glycosylation with tunicamycin decreased cell viability by 20%. In addition, tunicamycin inhibited the androgen-induced expression of AR target genes KLK3 and CaMKK2 by 50%. RTKs have been shown to enhance AR activity and we used an antibody array to identify changes in the phosphorylation status of RTKs in response to androgen stimulation. Hormone treatment increased the activity of Insulin like Growth Factor 1-Receptor (IGF-1R ten-fold and this was associated with a concomitant increase in the N-linked glycosylation of the receptor, analyzed by lectin enrichment experiments. Glycosylation is known to be important for the processing and stability of RTKs. Inhibition of N-linked glycosylation resulted in accumulation of IGF-1R pro-receptor with altered mobility as shown by immunoprecipitation. Confocal imaging revealed that androgen induced plasma-membrane localization of IGF-1R was blocked by tunicamycin. In conclusion we have established that the glycosylation of IGF-1R is necessary for the full activation of the receptor in response to androgen treatment and that perturbing this process can break the feedback loop between AR and IGF-1R activation in prostate cells. Achieving similar results selectively in a clinical setting will be an

  13. [Tyrosine-protein kinase activity in breast neoplasm. Comparison with activity obtained in benign diseases and in normal tissues].

    Science.gov (United States)

    Pierart, J; Oñate, E; Klaassen, R; Cid, L; Gutierrez, S; Talbot, E; Ross, E; Zambrano, C; Burmeister, R; Puchi, M

    1995-02-01

    Tyrosine protein kinase (TPK) activity is associated to malignant cellular transformation. This work compares TPK activity in 27 surgical biopsy samples of mammary carcinoma, 10 samples of fibroadenomas, 13 samples of fibrocystic breast disease and 27 samples of normal mammary tissue. TPK activity was determined in tissue homogenates using (Val5) angiotensin II as exogenous substrate. In samples of mammary carcinoma, TPK activity was 33.86 +/- 31.98 pmol P32/mg protein/30 min. This value was significantly higher that those observed in fibrocystic disease (3.92 +/- 2.35), fibroadenomas (13.86 +/- 10.9) and normal tissue (3.56 +/- 3.02).

  14. 8-THP-DHI analogs as potent Type I dual TIE-2/VEGF-R2 receptor tyrosine kinase inhibitors.

    Science.gov (United States)

    Hudkins, Robert L; Zulli, Allison L; Underiner, Ted L; Angeles, Thelma S; Aimone, Lisa D; Meyer, Sheryl L; Pauletti, Daniel; Chang, Hong; Fedorov, Elena V; Almo, Steven C; Fedorov, Alexander A; Ruggeri, Bruce A

    2010-06-01

    A novel series of 8-(2-tetrahydropyranyl)-12,13-dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazoles (THP-DHI) was synthesized and evaluated as dual TIE-2 and VEGF-R2 receptor tyrosine kinase inhibitors. Development of the structure-activity relationships (SAR) with the support of X-ray crystallography led to identification of 7f and 7g as potent, selective dual TIE-2/VEGF-R2 inhibitors with excellent cellular potency and acceptable pharmacokinetic properties. Compounds 7f and 7g were orally active in tumor models with no observed toxicity.

  15. Primary cilia and coordination of receptor tyrosine kinase (RTK) and transforming growth factor β (TGF-β) signaling

    DEFF Research Database (Denmark)

    Christensen, Søren Tvorup; Morthorst, Stine Kjær; Mogensen, Johanne Bay;

    2017-01-01

    are at the root of a pleiotropic group of diseases and syndromic disorders called ciliopathies. In this review, we present an overview of primary cilia-mediated regulation of receptor tyrosine kinase (RTK) and transforming growth factor β (TGF-β) signaling. Further, we discuss how defects in the coordination...... in their extracellular environment and integrate and transmit signaling information to the cell to regulate various cellular, developmental, and physiological processes. Many different signaling pathways have now been shown to rely on primary cilia to function properly, and mutations that lead to ciliary dysfunction...

  16. The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine kinase inhibitor tyrphostin 23

    DEFF Research Database (Denmark)

    Hohnholt, Michaela C; Blumrich, Eva-Maria; Waagepetersen, Helle S

    2017-01-01

    production. In addition, T23-treatment strongly increased the molecular carbon labeling of the TCA cycle intermediates citrate, succinate, fumarate and malate, and significantly increased the incorporation of (13)C-labelling into the amino acids glutamate, glutamine and aspartate. These results clearly......Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases and has been considered as potential anti-cancer drug. T23 was recently reported to acutely stimulate the glycolytic flux in primary cultured astrocytes. To investigate whether T23 also affects the tricarboxylic acid (TCA...

  17. Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

    OpenAIRE

    1994-01-01

    Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation...

  18. Salmonella induces SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling pathways in commercial and wild-type turkey leukocytes

    Science.gov (United States)

    Previous studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on d...

  19. Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway.

    Science.gov (United States)

    Marzec, M; Kasprzycka, M; Liu, X; El-Salem, M; Halasa, K; Raghunath, P N; Bucki, R; Wlodarski, P; Wasik, M A

    2007-08-16

    The mechanisms of cell transformation mediated by the nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) tyrosine kinase are only partially understood. Here, we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma display persistent activation of mammalian target of rapamycin (mTOR) as determined by phosphorylation of mTOR targets S6rp and 4E-binding protein 1 (4E-BP1). The mTOR activation is serum growth factor-independent but nutrient-dependent. It is also dependent on the expression and enzymatic activity of NPM/ALK as demonstrated by cell transfection with wild-type and functionally deficient NPM/ALK, small interfering RNA (siRNA)-mediated NPM/ALK depletion and kinase activity suppression using the inhibitor WHI-P154. The NPM/ALK-induced mTOR activation is transduced through the mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway and, to a much lesser degree, through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Accordingly, whereas the low-dose PI3K inhibitor wortmannin and Akt inhibitor III profoundly inhibited Akt phosphorylation, they had a very modest effect on S6rp and 4E-BP1 phosphorylation. In turn, MEK inhibitors U0126 and PD98059 and siRNA-mediated depletion of either ERK1 or ERK2 inhibited S6rp phosphorylation much more effectively. Finally, the mTOR inhibitor rapamycin markedly decreased proliferation and increased the apoptotic rate of ALK+TCL cells. These findings identify mTOR as a novel key target of NPM/ALK and suggest that mTOR inhibitors may prove effective in therapy of ALK-induced malignancies.

  20. CEP-701 and CEP-751 inhibit constitutively activated RET tyrosine kinase activity and block medullary thyroid carcinoma cell growth.

    Science.gov (United States)

    Strock, Christopher J; Park, Jong-In; Rosen, Mark; Dionne, Craig; Ruggeri, Bruce; Jones-Bolin, Susan; Denmeade, Samuel R; Ball, Douglas W; Nelkin, Barry D

    2003-09-01

    All of the cases of medullary thyroid carcinoma (MTC) express the RET receptor tyrosine kinase. In essentially all of the hereditary cases and approximately 40% of the sporadic cases of MTC, the RET kinase is constitutively activated by mutation. This suggests that RET may be an effective therapeutic target for treatment of MTC. We show that the indolocarbazole derivatives, CEP-701 and CEP-751, inhibit RET in MTC cells. These compounds effectively inhibit RET phosphorylation in a dose-dependent manner at concentrations <100 nM in 0.5% serum and at somewhat higher concentrations in the presence of 16% serum. They also blocked the growth of these MTC cells in culture. CEP-751 and its prodrug, CEP-2563, also inhibited tumor growth in MTC cell xenografts. These results show that inhibiting RET can block the growth of MTC cells and may have a therapeutic benefit in MTC.

  1. Proliferation of Ewing sarcoma cell lines is suppressed by the receptor tyrosine kinase inhibitors gefitinib and vandetanib

    Directory of Open Access Journals (Sweden)

    Åman Pierre

    2008-01-01

    Full Text Available Abstract Background Tyrosine kinase inhibitors (TKIs have gained much attention in recent years as targeted agents for the treatment of a wide range of human cancers. We have investigated the effect of the TKIs gefitinib and vandetanib on tumor cell lines derived from Ewing sarcoma, a highly malignant tumor affecting bone and soft tissue in children and young adults. Gefitinib is an inhibitor of epidermal growth factor receptor tyrosine kinase activity (EGFR and vandetanib selectively targets vascular endothelial growth factor receptor-2 (VEGFR-2 with additional activity against VEGFR-3, EGFR and RET kinase receptors. Results Two Ewing sarcoma cell lines investigated showed high levels of nuclear EGFR expression as well as moderate expression in plasma membrane and cytoplasm. When treated with concentrations of 5 μM and more of either gefitinib or vandetanib, we observed a significant decrease in cell proliferation. However, there were no detectable changes in p44/42 MAPK and Akt-1 phosphorylation, or in the expression of cyclin D1 or c-Myc following gefitinib or vandetanib treatment. Conclusion We conclude that Ewing sarcoma tumor cell proliferation is not highly sensitive to inhibition of EGFR signaling alone or the simultaneous inhibition of VEGFR receptors, EGFR and RET kinase. Decreased tumor cell proliferation could be achieved with gefitinib and vandetanib, but only at higher doses where non-specific effects of the compounds may be overriding. As Ewing tumor cells do not seem to depend on EGFR and VEGFR pathways for survival, other key factors in the cellular signaling of Ewing sarcoma should be targeted in order to obtain a potent therapeutic response.

  2. Regeneration of human epidermis on acellular dermis is impeded by small-molecule inhibitors of EGF receptor tyrosine kinase.

    Science.gov (United States)

    Forsberg, Sofi; Ostman, Arne; Rollman, Ola

    2008-10-01

    The family of human epidermal growth factor receptors (EGFR, HER2-4) exerts key functions in normal and malignant epithelial cells. Both EGFR and HER2 are valuable targets for anti-cancer drugs by interfering with ligand binding, receptor dimerization, or tyrosine kinase activity. A similar therapeutic strategy has been advocated for chronic psoriasis since plaque lesions overexpress EGFR and its ligands. Our aim was to characterize EGFR/HER2 protein expression in skin cultures and to evaluate the effects of tyrosine kinase inhibitors on epidermal outgrowth, morphology, and EGFR activation. Human skin explants were established on cell-free dermis and cultured at the air-liquid interface. The impact of small-molecule HER inhibitors on outgrowth was assayed by fluorescence-based image analysis and histometry. Effects of a dual EGFR/HER2 kinase inhibitor, PKI166, on neoepidermis were studied by immunohistochemistry and Western blot. Receptor immunostaining showed in vivo-like distributions with highest EGFR intensity in the proliferative layers whereas HER2 was mainly expressed by suprabasal keratinocytes. Reepithelialization was associated with EGFR autophosphorylation irrespective of exogenous ligand stimulation. PKI166 inhibited neoepidermal EGFR activation, keratinocyte proliferation, and outgrowth from normal and psoriatic skin explants. The rate of epidermalization in presence of other HER inhibitors varied suggesting that drug specificity, potency, and reversibility determine the dynamic outcome. Overall, agents predominantly targeting EGFR kinase were more efficient inhibitors of epidermal regeneration than an HER2-selective drug. The study illustrates the usefulness of a dynamic skin model and emphasizes the potential of HER-directed approaches to control epidermal growth in hyperproliferative skin disorders.

  3. Mice lacking protein tyrosine kinase fyn develop a T helper-type 1 response and resistLeishmania major infection.

    Science.gov (United States)

    Yamakami, K; Akao, S; Wakabayashi, K; Tadakuma, T; Yoshizawa, N

    2001-07-01

    Fyn is a Src family protein tyrosine kinase associated with TCR/CD3 complex. Fyn appears to play a role in the activation of T cells based on its enzymatic activation and tyrosine phosphorylation following the ligation of TCR/CD3, and it also plays a critical role in the calcium flux and interleukin-2 (IL-2) production. The protective response against murineLeishmania major infection is associated with the T helper-type 1 (Th1) responses and the ability to modulate Th1 cytokines such as IL-2 and interferon-γ, respectively. The role of Fyn tyrosine kinasein vivo was directly examined by the response to infection withL. major in C57BL/6fyn-deficient mice. Despite the absence of Fyn, the mice remained resistant to this infection with only mild lesion development, and, they demonstrated Th1 responses as assessed by the delayed-type hyper-sensitivity response and cytokine milieu. The findings in thefyn-deficient mice failed to support a relationship between the anticipated functions of Fynin vitro and the immune response toL. major infectionin vivo. As a result, in leishmanial disease, Fyn probably plays a minor role in the protective immune response and is, therefore, not a key factor in such a response.

  4. The 44 kDa Pim-1 kinase directly interacts with tyrosine kinase Etk/BMX and protects human prostate cancer cells from apoptosis induced by chemotherapeutic drugs.

    Science.gov (United States)

    Xie, Y; Xu, K; Dai, B; Guo, Z; Jiang, T; Chen, H; Qiu, Y

    2006-01-05

    Protein kinase Pim-1 has been implicated in the development of hematopoietic and prostatic malignancies. Here, we present the evidence that two isoforms, the 44 and 33 kDa Pim-1, are expressed in all human prostate cancer cell lines examined. The subcellular localization of human 44 kDa Pim-1 is primarily on the plasma membrane, while the 33 kDa isoform is present in both the cytosol and nucleus in PCA cells. The 44 kDa Pim-1 contains the proline-rich motif at the N-terminus and directly binds to the SH3 domain of tyrosine kinase Etk. Such interaction leads to the activation of Etk kinase activity possibly by competing with the tumor suppressor p53. This is corroborated by the fact that overexpression of the 44 kDa Pim-1 in prostate cancer cells confers the resistance to chemotherapeutic drugs. Our results suggest that these two isoforms of Pim-1 kinase may regulate distinct substrates and the 44 kDa Pim-1 may play a more prominent role in drug resistance in prostate cancer cells.

  5. The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response — Biological and Clinical Aspects

    Energy Technology Data Exchange (ETDEWEB)

    Medová, Michaela, E-mail: michaela.medova@dkf.unibe.ch; Aebersold, Daniel M.; Zimmer, Yitzhak, E-mail: michaela.medova@dkf.unibe.ch [Department of Radiation Oncology, Inselspital, Bern University Hospital, and University of Bern, 3010 Bern (Switzerland); Department of Clinical Research, University of Bern, DKF, MEM-E807, Murtenstrasse 35, 3010 Bern (Switzerland)

    2013-12-19

    Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.

  6. Bmx, a member of the Tec family of nonreceptor tyrosine kinases, is a novel participant in pharmacological cardioprotection.

    Science.gov (United States)

    Zhang, Jun; Ping, Peipei; Wang, Guang-Wu; Lu, Ming; Pantaleon, Dawn; Tang, Xian-Liang; Bolli, Roberto; Vondriska, Thomas M

    2004-11-01

    Previous studies have indicated that PKC-epsilon is a central regulator of protective signal transduction in the heart. However, the signaling modules through which PKC-epsilon exerts its protective effects have only begun to be understood. We have identified a novel participant in the PKC-epsilon signaling system in cardioprotection, the nonreceptor tyrosine kinase Bmx. Functional proteomic analyses of PKC-epsilon signaling complexes identified Bmx as a member of these complexes. Subsequent studies in rabbits have indicated that Bmx is activated by nitric oxide (NO) in the heart, concomitant with the late phase of NO donor-induced protection, and provide the first analysis of Bmx expression/distribution in the setting of cardioprotection. In addition, increased expression of Bmx induced by NO donors was blocked by the same mechanism that blocked cardioprotection: inhibition of PKC with chelerythrine. These findings indicate that a novel type of PKC-tyrosine kinase module (involving Bmx) is formed in the heart and may be involved in pharmacological cardioprotection by NO donors.

  7. Coexpression of receptor-tyrosine-kinases in gastric adenocarcinoma-a rationale for a molecular targeting strategy?

    Institute of Scientific and Technical Information of China (English)

    Daniel Drescher; Thomas Wehler; Andreas Teufel; Kerstin Herzer; Thomas Fischer; Martin R Berger; Theodor Junginger; Peter R Galle; Carl C Schimanski; Markus Moehler; Ines Gockel; Kirsten Frerichs; Annett Müller; Friedrich Dünschede; Thomas Borschitz; Stefan Biesterfeld; Martin Holtmann

    2007-01-01

    AIM: To define the (co-)expression pattern of target receptor-tyrosine-kinases (RTK) in human gastric adenocarcinoma.METHODS: The (co-)expression pattern of VEGFR1-3,PDGFRα/β and EGFR1 was analyzed by RT-PCR in 51 human gastric adenocarcinomas. In addition, IHC staining was applied for confirmation of expression and analysis of RTK Iocalisation.RESULTS: The majority of samples revealed a VEGFR1(98%), VEGFR2 (80%), VEGFR3 (67%), PDGFRα(82%) and PDGFRβ (82%) expression, whereas only 62% exhibited an EGFR1 expression. 78% of cancers expressed at least four out of six RTKs. While VEGFR1-3and PDGFRα revealed a predominantly cytoplasmatic staining in tumor cells, accompanied by an additional nuclear staining for VEGFR3, EGFR1 was almost exclusively detected on the membrane of tumor cells.PDGFRβ was restricted to stromal pericytes, which also depicted a PDGFRα expression.CONCLUSION: Our results reveal a high rate of receptor-tyrosine-kinases coexpression in gastric adenocarcinoma and might therefore encourage an application of multiple-target RTK-inhibitors within a combination therapy.

  8. A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Park, So Jung; Park, Young Jun; Shin, Ji Hyun; Kim, Eun Sung [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of); Hwang, Jung Jin; Jin, Dong-Hoon; Kim, Jin Cheon [Institute for Innovative Cancer Research, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Cho, Dong-Hyung, E-mail: dhcho@khu.ac.kr [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of)

    2011-05-13

    Highlights: {yields} We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. {yields} Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. {yields} Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. {yields} Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

  9. A positive feedback loop between Gli1 and tyrosine kinase Hck amplifies shh signaling activities in medulloblastoma.

    Science.gov (United States)

    Shi, X; Zhan, X; Wu, J

    2015-11-30

    Sonic hedgehog (Shh) signaling is critical during normal development, and the abnormal activation of the Shh pathway is involved in many human cancers. As a target gene of the Shh pathway and as a transcription activator downstream of Shh signaling, Gli1 autoregulates and increases Shh signaling output. Gli1 is one of the key oncogenic factors in Shh-induced tumors such as medulloblastoma. Gli1 is posttranslationally modified, but the nature of the active form of Gli1 was unclear. Here we identified a Src family kinase Hck as a novel activator of Gli1. In Shh-responsive NIH3T3 cells, Hck interacts with Gli1 and phosphorylates multiple tyrosine residues in Gli1. Gli1-mediated target gene activation was significantly enhanced by Hck with both kinase activity-dependent and -independent mechanisms. We provide evidence showing that Hck disrupts the interaction between Gli1 and its inhibitor Sufu. In both NIH3T3 cells and cerebellum granule neuron precursors, the Hck gene is also a direct target of Gli1. Therefore, Gli1 and Hck form a positive feedback loop that amplifies Shh signaling transcription outcomes. In Shh-induced medulloblastoma, Hck is highly expressed and Gli1 is tyrosine phosphorylated, which may enhance the tumorigenic effects of the Gli1 oncogene. RNAi-mediated inhibition of Hck expression significantly repressed medulloblastoma cell growth. In summary, a novel positive feedback loop contributes to maximal Gli1 oncogenic activities in Shh-induced tumors such as medulloblastoma.

  10. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp; Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto, E-mail: nyama@faculty.chiba-u.jp

    2014-09-26

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

  11. Inhaled tyrosine kinase inhibitors for pulmonary hypertension: a possible future treatment

    Directory of Open Access Journals (Sweden)

    Pitsiou G

    2014-10-01

    Full Text Available Georgia Pitsiou,1 Paul Zarogoulidis,1 Dimitris Petridis,2 Ioannis Kioumis,1 Sofia Lampaki,1 John Organtzis,1 Konstantinos Porpodis,1 Antonis Papaiwannou,1 Theodora Tsiouda,3 Wolfgang Hohenforst-Schmidt,4 Stylianos Kakolyris,5 Konstantinos Syrigos,6 Haidong Huang,7 Qiang Li,7 J Francis Turner,8 Konstantinos Zarogoulidis1 1Pulmonary Department, G Papanikolaou General Hospital, Aristotle University of Thessaloniki, 2Department of Food Technology, School of Food Technology and Nutrition, Alexander Technological Educational Institute, 3Internal Medicine Department, Thegenio Anticancer Hospital, Thessaloniki, Greece; 4II Medical Department, Coburg Regional Hospital, Coburg, Germany; 5Oncology Department, Sotiria Hospital of Chest Diseases, University of Athens, Athens, 6Oncology Department, University General Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis, Greece; 7Department of Respiratory Diseases, Changhai Hospital/First Affiliated Hospital of the Second Military Medical University, Shanghai, People’s Republic of China; 8Division of Interventional Pulmonology and Medical Oncology, Cancer Treatment Centers of America, Western Regional Medical Center, Goodyear, AZ, USA Abstract: Pulmonary hypertension is a disease with severe consequences for the human body. There are several diseases and situations that induce pulmonary hypertension and are usually underdiagnosed. Treatments include conventional medical therapies and oral, inhaled, intravenous, and subcutaneous options. Depending on its severity, heart or lung transplant may also be an option. A possible novel treatment could be tyrosine kinase inhibitors. We conducted experiments with three jet nebulizers and three ultrasound nebulizers with erlotinib, gefitinib, and imatinib. Different residual cup designs and residual cup loadings were used in order to identify the best combination to produce droplets of less than 5 µm in mass median aerodynamic diameter. We

  12. rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

    Science.gov (United States)

    Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J

    1994-04-08

    We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

  13. ABL tyrosine kinase inhibition variable effects on the invasive properties of different triple negative breast cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Clément Chevalier

    Full Text Available The non-receptor tyrosine kinase ABL drives myeloid progenitor expansion in human chronic myeloid leukemia. ABL inhibition by the tyrosine kinase inhibitor nilotinib is a first-line treatment for this disease. Recently, ABL has also been implicated in the transforming properties of solid tumors, including triple negative (TN breast cancer. TN breast cancers are highly metastatic and several cell lines derived from these tumors display high invasive activity in vitro. This feature is associated with the activation of actin-rich membrane structures called invadopodia that promote extracellular matrix degradation. Here, we investigated nilotinib effect on the invasive and migratory properties of different TN breast cancer cell lines. Nilotinib decreased both matrix degradation and invasion in the TN breast cancer cell lines MDA-MB 231 and MDA-MB 468. However, and unexpectedly, nilotinib increased by two-fold the invasive properties of the TN breast cancer cell line BT-549 and of Src-transformed fibroblasts. Both display much higher levels of ABL kinase activity compared to MDA-MB 231. Similar effects were obtained by siRNA-mediated down-regulation of ABL expression, confirming ABL central role in this process. ABL anti-tumor effect in BT-549 cells and Src-transformed fibroblasts was not dependent on EGF secretion, as recently reported in neck and squamous carcinoma cells. Rather, we identified the TRIO-RAC1 axis as an important downstream element of ABL activity in these cancer cells. In conclusion, the observation that TN breast cancer cell lines respond differently to ABL inhibitors could have implications for future therapies.

  14. Os inibidores de tirosino quinase de segunda geração The inhibitors of tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Márcia T. Delamain

    2008-04-01

    Full Text Available O imatinibe tem sido confirmado como terapia de primeira linha para a Leucemia Mielóide Crônica (LMC por apresentar respostas duradouras na maior parte dos pacientes, principalmente nos que se encontram em fase precoce da doença. Entretanto, resistência ou intolerância ao imatinibe podem ocorrer. A resistência ao imatinibe ocorre com muito mais freqüência em fases mais avançadas da doença, sendo a causa mais comum o desenvolvimento de mutações no sítio BCR-ABL. Em face deste problema, novos inibidores de tirosino quinase têm sido desenvolvidos, com maior potência, diminuindo assim a chance de desenvolvimento de resistência ao tratamento. O nilotinibe e o dasatinibe são dois exemplos de inibidores de segunda geração de tirosino quinase recentemente aprovados. Ambos têm demonstrado excelentes resultados em pacientes que desenvolvem resistência ou são intolerantes ao imatinibe.Despite the success with imatinib as the first choice treatment of chronic myeloid leukemia (CML, there is still a subset of patients that do not respond optimally to or are intolerant of this drug or lose response. Imatinib resistance can occur at any phase, but it is more frequent in advanced phases, with mutations in the BCR-ABL kinase domain being the most common mechanism of resistance. More potent tyrosine kinase inhibitors have been developed that can overcome resistance to imatinib. Nilotinib and dasatinib are good examples of new tyrosine kinase inhibitors that are available. With these new agents, patients who develop imatinib resistance or those unable to tolerate imatinib treatment can achieve significant clinical responses.

  15. ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia.

    Science.gov (United States)

    Shankar, Deepa B; Li, Junling; Tapang, Paul; Owen McCall, J; Pease, Lori J; Dai, Yujia; Wei, Ru-Qi; Albert, Daniel H; Bouska, Jennifer J; Osterling, Donald J; Guo, Jun; Marcotte, Patrick A; Johnson, Eric F; Soni, Niru; Hartandi, Kresna; Michaelides, Michael R; Davidsen, Steven K; Priceman, Saul J; Chang, Jenny C; Rhodes, Katrin; Shah, Neil; Moore, Theodore B; Sakamoto, Kathleen M; Glaser, Keith B

    2007-04-15

    In 15% to 30% of patients with acute myeloid leukemia (AML), aberrant proliferation is a consequence of a juxtamembrane mutation in the FLT3 gene (FMS-like tyrosine kinase 3-internal tandem duplication [FLT3-ITD]), causing constitutive kinase activity. ABT-869 (a multitargeted receptor tyrosine kinase inhibitor) inhibited the phosphorylation of FLT3, STAT5, and ERK, as well as Pim-1 expression in MV-4-11 and MOLM-13 cells (IC(50) approximately 1-10 nM) harboring the FLT3-ITD. ABT-869 inhibited the proliferation of these cells (IC(50) = 4 and 6 nM, respectively) through the induction of apoptosis (increased sub-G(0)/G(1) phase, caspase activation, and PARP cleavage), whereas cells harboring wild-type (wt)-FLT3 were less sensitive. In normal human blood spiked with AML cells, ABT-869 inhibited phosphorylation of FLT3 (IC(50) approximately 100 nM), STAT5, and ERK, and decreased Pim-1 expression. In methylcellulose-based colony-forming assays, ABT-869 had no significant effect up to 1000 nM on normal hematopoietic progenitor cells, whereas in AML patient samples harboring both FLT3-ITD and wt-FLT3, ABT-869 inhibited colony formation (IC(50) = 100 and 1000 nM, respectively). ABT-869 dose-dependently inhibited MV-4-11 and MOLM-13 flank tumor growth, prevented tumor formation, regressed established MV-4-11 xenografts, and increased survival by 20 weeks in an MV-4-11 engraftment model. In tumors, ABT-869 inhibited FLT3 phosphorylation, induced apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]) and decreased proliferation (Ki67). ABT-869 is under clinical development for AML.

  16. Structural Characterization of Proline-rich Tyrosine Kinase 2 (PYK2) Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design

    Energy Technology Data Exchange (ETDEWEB)

    Han, Seungil; Mistry, Anil; Chang, Jeanne S.; Cunningham, David; Griffor, Matt; Bonnette, Peter C.; Wang, Hong; Chrunyk, Boris A.; Aspnes, Gary E.; Walker, Daniel P.; Brosius, Arthur D.; Buckbinder, Leonard; Pfizer

    2009-05-21

    Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.

  17. Identification of AKN-032, a novel 2-aminopyrazine tyrosine kinase inhibitor, with significant preclinical activity in acute myeloid leukemia.

    Science.gov (United States)

    Eriksson, Anna; Höglund, Martin; Lindhagen, Elin; Aleskog, Anna; Hassan, Sadia Bashir; Ekholm, Carina; Fhölenhag, Karin; Jensen, Annika Jenmalm; Löthgren, Agneta; Scobie, Martin; Larsson, Rolf; Parrow, Vendela

    2010-11-15

    Aberrant signal transduction by mutant or overexpressed protein kinases has emerged as a promising target for treatment of acute myeloid leukemia (AML). We here present a novel low molecular weight kinase inhibitor, AKN-032, targeting the FMS-like tyrosine kinase 3 (FLT3) and discovered in a new type of screening funnel combining the target therapy approach with sequential cellular screens. AKN-032 was identified among 150 selected hits from three different high throughput kinase screens. Further characterization showed inhibitory activity on FLT3 enzyme with an IC(50) of 70 nM. Western blot analysis revealed reduced autophosphorylation of the FLT3-receptor in AML cell line MV4-11 cells after exposure to AKN-032. Flow cytometry disclosed cytotoxic activity against MV4-11, but not against non-malignant 3T3-L1 fibroblast cells. Using a fluorometric microculture cytotoxicity assay, AKN-032 was tested against 15 cell lines and displayed a potent cytotoxic activity in AML cell lines MV4-11 (IC(50)=0.4 μM) and Kasumi-1 (IC(50)=2.3 μM). AKN-032 was also highly cytotoxic in tumor cells from AML patients in vitro. Furthermore, AKN-032 demonstrated significant antileukemic effect in a relatively resistant in vivo hollow fiber mouse model. No major toxicity was observed in the animals. In conclusion, AKN-032 is a promising new kinase inhibitor with significant in vivo and in vitro activity in AML. Results from the hollow fiber mouse assay suggest a favorable toxicity profile. Future studies will focus on pharmacokinetic properties, toxicity as well as further clarifying the mechanisms of action of AKN-032 in AML.

  18. Activation of extracellular signal-regulated kinase during silibinin-protected, isoproterenol-induced apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent

    Institute of Scientific and Technical Information of China (English)

    Bei ZHOU; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Fumiaki UCHIUMI; Takashi IKEJIMA

    2007-01-01

    Aim: To investigate the mechanism of silibinin-protected isoproterenol-induced apoptosis in rat cardiac myocytes.Methods: The viability of rat cardiac myocytes was measured by MTT method. The apoptotic ratio was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Protein kinase C (PKC) activity assay was carried out according to the instructions of the PepTag non-radioactive protein kinase C assay kit. Western blot analysis was used to evaluate the level of Ras, Raf-1 and mitogen-activated protein kinase (MAPK) expression.Results: The protective effects of silibinin were significantly sup-pressed by inhibitors, including genistein, manumycin A and GW5074 [inhibitors for protein tyrosine kinases (PTK), Ras and Raf- 1, respectively]. The exposure of rat cardiac myocytes to isoproterenol alone caused decreased PKC activity, which was prevented by pretreatment with silibinin dose-dependently. Simultaneously,the increased expression of Ras and Raf-1 activated by silibinin were blocked by the PKC inhibitor, stauroporine. In addition, the extracellularly responsive kinase (ERK) inhibitor, PD98059, suppressed silibinin-protected apoptosis, whereas the p38 MAPK inhibitor, SB203580, protected cardiac myocytes from isoproterenol-induced injury, and the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 had no protective effects. Furthermore, Western blot analysis showed that the expres-sion of phosphorylated ERK was increased by silibinin, the expression of phos-phorylated p38 MAPK was decreased and total ERK, p38, JNK and phosphory-lated JNK MAPK did not change after treatment with both isoproterenol and silibinin. Furthermore, pretreatment of cardiac myocyte with PKC, Ras and Raf inhibitors significantly blocked ERK phosphorylation.Conclusion: Silibinin is suggested to protect isoproterenol-induced rat cardiac myocyte apoptosis by activating the tyrosine kinase pathway, PKC and MAPK pathways.

  19. Specific oncogenic activity of the Src-family tyrosine kinase c-Yes in colon carcinoma cells.

    Science.gov (United States)

    Sancier, Florence; Dumont, Aurélie; Sirvent, Audrey; Paquay de Plater, Ludmilla; Edmonds, Thomas; David, Géraldine; Jan, Michel; de Montrion, Catherine; Cogé, Francis; Léonce, Stéphane; Burbridge, Michael; Bruno, Alain; Boutin, Jean A; Lockhart, Brian; Roche, Serge; Cruzalegui, Francisco

    2011-02-24

    c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.

  20. Bi-directional regulation between tyrosine kinase Etk/BMX and tumor suppressor p53 in response to DNA damage.

    Science.gov (United States)

    Jiang, Tianyun; Guo, Zhiyong; Dai, Bojie; Kang, Miyoung; Ann, David K; Kung, Hsing-Jien; Qiu, Yun

    2004-11-26

    Etk/Bmx, a member of the Tec family of nonreceptor tyrosine kinases, has been implicated in the regulation of various cellular processes including proliferation, differentiation, motility, and apoptosis. Here, we report the identification of Tec family kinases as the potential interacting proteins of the tumor suppressor p53 by an Src homology 3 domain array screening. Etk is physically associated with p53 through its Src homology 3 domain and the proline-rich domain of p53. Induction of p53 expression by DNA damage inhibits Etk activity in several cell types. Down-regulation of Etk expression by a specific small interfering RNA sensitizes prostate cancer cells to doxorubicin-induced apoptosis, suggesting that inhibition of Etk activity is required for apoptosis in response to DNA damage. We also show that Etk primarily interacts with p53 in the cytoplasm and that such interaction leads to bidirectional inhibition of the activities of both proteins. Overexpression of Etk in prostate cancer cells results in inhibition of p53 transcriptional activity and its interaction with the mitochondrial protein BAK and confers the resistance to doxorubicin. Therefore, we propose that the stoichiometry between p53 and the Tec family kinases in a given cell type may determine its sensitivity to chemotherapeutic drugs.

  1. Patients harboring EGFR mutation after primary resistance to crizotinib and response to EGFR-tyrosine kinase inhibitor

    Directory of Open Access Journals (Sweden)

    Wang WX

    2016-01-01

    Full Text Available Wenxian Wang,1 Xiaowen Jiang,1 Zhengbo Song,1,2 Yiping Zhang1,2 1Department of Chemotherapy, Zhejiang Cancer Hospital, 2Key Laboratory Diagnosis and Treatment Technology on Thoracic Oncology, Hangzhou, Zhejiang, People’s Republic of China Abstract: Anaplastic lymphoma kinase (ALK rearrangement lung cancer responds to ALK tyrosine kinase inhibitors. It is known that many cases ultimately acquired resistance to crizotinib. However, a case of primary resistance is rare. We present a case of harboring exon 19 deletion in epidermal growth factor receptor in ALK rearranged lung adenocarcinoma, who experienced a partial tumor response to icotinib after failure with crizotinib therapy and chemotherapy. Considering the partial response, we conclude that it is important to find the cause of resistance to crizotinib. We detected gene mutations with plasma by the next-generation sequencing; the next-generation sequencing demonstrates an attractive system to identify mutations improving the outcome of patients with a deadly disease. Keywords: non-small cell lung cancer, anaplastic lymphoma kinase, crizotinib, epidermal growth factor receptor

  2. Lipid raft localization of EGFR alters the response of cancer cells to the EGFR tyrosine kinase inhibitor gefitinib.

    Science.gov (United States)

    Irwin, Mary E; Mueller, Kelly L; Bohin, Natacha; Ge, Yubin; Boerner, Julie L

    2011-09-01

    Epidermal growth factor receptor (EGFR) is overexpressed in many cancer types including ~30% of breast cancers. Several small molecule tyrosine kinase inhibitors (TKIs) targeting EGFR have shown clinical efficacy in lung and colon cancers, but no benefit has been noted in breast cancer. Thirteen EGFR expressing breast cancer cell lines were analyzed for response to EGFR TKIs. Seven were found to be EGFR TKI resistant; while shRNA knockdown of EGFR determined that four of these cell lines retained the requirement of EGFR protein expression for growth. Interestingly, EGFR localized to plasma membrane lipid rafts in all four of these EGFR TKI-resistant cell lines, as determined by biochemical raft isolation and immunofluorescence. When lipid rafts were depleted of cholesterol using lovastatin, all four cell lines were sensitized to EGFR TKIs. In fact, the effects of the cholesterol biosynthesis inhibitors and gefitinib were synergistic. While gefitinib effectively abrogated phosphorylation of Akt- and mitogen-activated protein kinase in an EGFR TKI-sensitive cell line, phosphorylation of Akt persisted in two EGFR TKI-resistant cell lines, however, this phosphorylation was abrogated by lovastatin treatment. Thus, we have shown that lipid raft localization of EGFR correlates with resistance to EGFR TKI-induced growth inhibition and pharmacological depletion of cholesterol from lipid rafts decreases this resistance in breast cancer cell lines. Furthermore, we have presented evidence to suggest that when EGFR localizes to lipid rafts, these rafts provide a platform to facilitate activation of Akt signaling in the absence of EGFR kinase activity.

  3. The NPM-ALK tyrosine kinase mimics TCR signalling pathways, inducing NFAT and AP-1 by RAS-dependent mechanisms.

    Science.gov (United States)

    Turner, Suzanne D; Yeung, Debra; Hadfield, Kathryn; Cook, Simon J; Alexander, Denis R

    2007-04-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expression is associated with the lymphoid malignancy anaplastic large cell lymphoma (ALCL) and results from a t(2;5) chromosomal translocation. We show that NPM-ALK induces Ras activation and phosphorylation of the ERK MAP Kinase consistent with activation of the Ras-MAP Kinase pathway. Furthermore, we demonstrate that activation of Ras is necessary for inducing transcription via NFAT/AP-1 composite transcriptional binding sites. This activity is dependent on NPM-ALK forming complexes with proteins that bind to autophosphorylated tyrosine residues at positions 156, 567 and 664, associated with binding to IRS-1, Shc and PLCgamma, respectively. Specifically, NPM-ALK activates transcription from the TRE promoter element, an AP-1 binding region, an activity dependent on both Ras and Shc activity. Our results show that NPM-ALK mimics activated T-cell receptor signalling by inducing pathways associated with the activation of NFAT/AP-1 transcription factors that bind to promoter elements found in a broad array of cytokine genes.

  4. A "liaison dangereuse" between AUF1/hnRNPD and the oncogenic tyrosine kinase NPM-ALK.

    Science.gov (United States)

    Fawal, Mohamad; Armstrong, Florence; Ollier, Severine; Dupont, Henri; Touriol, Christian; Monsarrat, Bernard; Delsol, Georges; Payrastre, Bernard; Morello, Dominique

    2006-10-15

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a chimeric protein expressed in a subset of cases of anaplastic large cell lymphoma (ALCL) for which constitutive expression represents a key oncogenic event. The ALK signaling pathway is complex and probably involves functional redundancy between various signaling substrates of ALK. Despite numerous studies on signaling mediators, the molecular mechanisms contributing to the distinct oncogenic features of NPM-ALK remain incompletely understood. The search for additional interacting partners of NPM-ALK led to the discovery of AUF1/hnRNPD, a protein implicated in AU-rich element (ARE)-directed mRNA decay. AUF1 was immunoprecipitated with ALK both in ALCL-derived cells and in NIH3T3 cells stably expressing NPM-ALK or other X-ALK fusion proteins. AUF1 and NPM-ALK were found concentrated in the same cytoplasmic foci, whose formation required NPM-ALK tyrosine kinase activity. AUF1 was phosphorylated by ALK in vitro and was hyperphosphorylated in NPM-ALK-expressing cells. Its hyperphosphorylation was correlated with increased stability of several AUF1 target mRNAs encoding key regulators of cell proliferation and with increased cell survival after transcriptional arrest. Thus, AUF1 could function in a novel pathway mediating the oncogenic effects of NPM-ALK. Our data establish an important link between oncogenic kinases and mRNA turnover, which could constitute a critical aspect of tumorigenesis.

  5. Bisubstrate analog probes for the insulin receptor protein tyrosine kinase: molecular yardsticks for analyzing catalytic mechanism and inhibitor design.

    Science.gov (United States)

    Hines, Aliya C; Parang, Keykavous; Kohanski, Ronald A; Hubbard, Stevan R; Cole, Philip A

    2005-08-01

    Bisubstrate analogs have the potential to provide enhanced specificity for protein kinase inhibition and tools to understand catalytic mechanism. Previous efforts led to the design of a peptide-ATP conjugate bisubstrate analog utilizing aminophenylalanine in place of tyrosine and a thioacetyl linker to the gamma-phosphate of ATP which was a potent inhibitor of the insulin receptor kinase (IRK). In this study, we have examined the contributions of various electrostatic and structural elements in the bisubstrate analog to IRK binding affinity. Three types of changes (seven specific analogs in all) were introduced: a Tyr isostere of the previous aminophenylalanine moiety, modifications of the spacer between the adenine and the peptide, and deletions and substitutions within the peptide moiety. These studies allowed a direct evaluation of the hydrogen bond strength between the anilino nitrogen of the bisubstrate analog and the enzyme catalytic base Asp and showed that it contributes 2.5 kcal/mol of binding energy, in good agreement with previous predictions. Modifications of the linker length resulted in weakened inhibitory affinity, consistent with the geometric requirements of an enzyme-catalyzed dissociative transition state. Alterations in the peptide motif generally led to diminished inhibitory potency, and only some of these effects could be rationalized based on prior kinetic and structural studies. Taken together, these results suggest that a combination of mechanism-based design and empirical synthetic manipulation will be necessary in producing optimized protein kinase bisubstrate analog inhibitors.

  6. Large, detergent-resistant complexes containing murine antigens Thy-1 and Ly-6 and protein tyrosine kinase p56lck.

    Science.gov (United States)

    Bohuslav, J; Cinek, T; Horejsí, V

    1993-04-01

    A number of human and mouse leukocyte surface (glyco)proteins anchored in a membrane via glycosylphosphatidylinositol (GPI) moiety have been previously shown to be noncovalently associated with protein tyrosine kinases (Science 1991. 254: 1016; J. Biol. Chem. 1992. 267: 12317). Here we show that two murine antigens of this group, Thy-1 and Ly-6, implicated in the activation of the T cells, are associated with each other, with the kinase p56lck and with several of potential kinase substrates in very large, detergent-resistant complexes, the size of which is between 50 and 200 nm, as determined by ultrafiltration and gel chromatography. Experiments on simultaneous solubilization of mixed human and mouse cells rule out that the observed complexes are artifacts induced by the detergent. Complexes of similar composition and properties were obtained when either detergents Brij-58, Nonidet-P40 or 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propane-sulfonate (Chaps) were used for solubilization of the cells, while octylglucoside at least partially dissociated them. These "GPI-complexes" may be essential for the well-known signal-transducing capacity of Thy-1 and Ly-6.

  7. A fully automated two-step synthesis of an {sup 18}F-labelled tyrosine kinase inhibitor for EGFR kinase activity imaging in tumors

    Energy Technology Data Exchange (ETDEWEB)

    Kobus, D.; Giesen, Y.; Ullrich, R.; Backes, H. [Max Planck Institute for Neurological Research with Klaus-Joachim-Zuelch Laboratories of the Max Planck Society and the Faculty of Medicine of the University of Cologne, Cologne (Germany); Neumaier, B. [Max Planck Institute for Neurological Research with Klaus-Joachim-Zuelch Laboratories of the Max Planck Society and the Faculty of Medicine of the University of Cologne, Cologne (Germany)], E-mail: bernd.neumaier@nf.mpg.de

    2009-11-15

    Radiolabelled epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitors potentially facilitate the assessment of EGFR overexpression in tumors. Since elaborate multi-step radiosyntheses are required for {sup 18}F-labelling of EGFR-specific anilinoquinazolines we report on the development of a two-step click labelling approach that was adapted to a fully automated synthesis module. 6-(4-N,N-Dimethylaminocrotonyl)amido-4-(3-chloro-4-fluoro)phenylamino-7-{l_brace}3- [4-(2-[{sup 18}F]fluoroethyl)-2,3,4-triazol-1-yl]propoxy{r_brace}quinazoline ([{sup 18}F]6) was synthesized via Huisgen 1,3-dipolar cycloaddition between 2-[{sup 18}F]fluoroethylazide ([{sup 18}F]4) and the alkyne modified anilinoquinazoline precursor 5. PET images of PC9 tumor xenograft using the novel biomarker showed promising results to visualize EGFR overexpression.

  8. Prevention of pulmonary vascular and myocardial remodeling by the combined tyrosine and serine-/threonine kinase inhibitor, sorafenib, in pulmonary hypertension and right heart failure

    Directory of Open Access Journals (Sweden)

    M. Klein

    2008-06-01

    Full Text Available Inhibition of tyrosine kinases can reverse pulmonary hypertension but little is known about the role of serine-/threonine kinases in vascular and myocardial remodeling. We investigated the effects of sorafenib, an inhibitor of the tyrosine kinases VEGFR, PDGFR and c-kit as well as the serine-/threonine kinase Raf-1, in pulmonary hypertension and right ventricular (RV pressure overload. In monocrotaline treated rats, sorafenib (10 mg·kg–1·d–1 p.o. reduced pulmonary arterial pressure, pulmonary artery muscularization and RV hypertrophy, and improved systemic hemodynamics (table 1. Sorafenib prevented phosphorylation of Raf-1 and suppressed activation of downstream signaling pathways (Erk 1/2. After pulmonary banding, sorafenib, but not the PDGFR/c-KIT/ABL-inhibitor imatinib reduced RV mass and RV filling pressure significantly. Congruent with these results, sorafenib only prevented ERK phosphorylation and vasopressin induced hypertrophy of the cardiomyocyte cell line H9c2 dose dependently (IC50 = 300 nM. Combined inhibition of tyrosine and serine-/threonine kinases by sorafenib prevents vascular and cardiac remodeling in pulmonary hypertension, which is partly mediated via inhibition of the Raf kinase pathway.

  9. Bmx tyrosine kinase regulates TLR4-induced IL-6 production in human macrophages independently of p38 MAPK and NFkapp}B activity.

    Science.gov (United States)

    Palmer, Christine D; Mutch, Brenda E; Workman, Sarita; McDaid, John P; Horwood, Nicole J; Foxwell, Brian M J

    2008-02-15

    Chronic inflammation, as seen in conditions such as rheumatoid arthritis and Crohn disease, is in part driven by discordant production of inflammatory cytokines, such as tumor necrosis factor-alpha and interleukin-6 (IL-6). Tyrosine kinase activity is essential to lipopolysaccharide-induced cytokine production in monocytes, and previous studies by us and others have implicated a role for the Tec kinase Bruton's tyrosine kinase (Btk) in inflammatory cytokine production. Here we show that knockdown of Btk using RNA interference results in decreased tumor necrosis factor-alpha, but not IL-6 production. Further investigations into the signaling mechanisms regulating IL-6 production led to the discovery that the Tec kinase bone marrow tyrosine kinase gene in chromosome X (Bmx) regulates Toll-like receptor-induced IL-6 production. Our data further showed that Bmx-dependent super-induction of IL-6 does not involve nuclear factor-kappaB activity. More detailed investigations of pathways downstream of Bmx signaling revealed that Bmx targets the IL-6 3' untranslated region to increase mRNA stabilization via a novel, thus far undefined, p38 mitogen activated protein kinase-independent pathway. These data have important implications for the design of therapeutics targeted against specific cytokines and their regulators in inflammatory disease.

  10. Neuropeptide-induced androgen independence in prostate cancer cells: roles of nonreceptor tyrosine kinases Etk/Bmx, Src, and focal adhesion kinase.

    Science.gov (United States)

    Lee, L F; Guan, J; Qiu, Y; Kung, H J

    2001-12-01

    The bombesin/gastrin-releasing peptide (GRP) family of neuropeptides has been implicated in various in vitro and in vivo models of human malignancies including prostate cancers. It was previously shown that bombesin and/or neurotensin (NT) acts as a survival and migratory factor(s) for androgen-independent prostate cancers. However, a role in the transition from an androgen-dependent to -refractory state has not been addressed. In this study, we investigate the biological effects and signal pathways of bombesin and NT on LNCaP, a prostate cancer cell line which requires androgen for growth. We show that both neurotrophic factors can induce LNCaP growth in the absence of androgen. Concurrent transactivation of reporter genes driven by the prostate-specific antigen promoter or a promoter carrying an androgen-responsive element (ARE) indicate that growth stimulation is accompanied by androgen receptor (AR) activation. Furthermore, neurotrophic factor-induced gene activation was also present in PC3 cells transfected with the AR but not in the parental line which lacks the AR. Given that bombesin does not directly bind to the AR and is known to engage a G-protein-coupled receptor, we investigated downstream signaling events that could possibly interact with the AR pathway. We found that three nonreceptor tyrosine kinases, focal adhesion kinase (FAK), Src, and Etk/BMX play important parts in this process. Etk/Bmx activation requires FAK and Src and is critical for neurotrophic factor-induced growth, as LNCaP cells transfected with a dominant-negative Etk/BMX fail to respond to bombesin. Etk's activation requires FAK, Src, but not phosphatidylinositol 3-kinase. Likewise, bombesin-induced AR activation is inhibited by the dominant-negative mutant of either Src or FAK. Thus, in addition to defining a new G-protein pathway, this report makes the following points regarding prostate cancer. (i) Neurotrophic factors can activate the AR, thus circumventing the normal growth

  11. Kinase activation of the non-receptor tyrosine kinase Etk/BMX alone is sufficient to transactivate STAT-mediated gene expression in salivary and lung epithelial cells.

    Science.gov (United States)

    Wen, X; Lin, H H; Shih, H M; Kung, H J; Ann, D K

    1999-12-31

    Etk/BMX is a non-receptor protein tyrosine kinase that requires a functional phosphatidylinositol 3-kinase via the pleckstrin homology domain to be activated by cytokine. In the present study, a conditionally active form of Etk was constructed by fusing the hormone-binding domain of estrogen receptor (ER) to an amino terminus truncated form of Etk, PHDelta1-68Etk, to generate DeltaEtk:ER. In stably transfected Pa-4DeltaEtk:ER cells, the activity of DeltaEtk:ER was stimulated within minutes by the treatment of DeltaEtk:ER stimulant, estradiol, and sustained for greater than 24 h. A robust induction in the phosphorylation of signal transducers and activators of transcription (STAT) proteins, including STAT1, STAT3, and STAT5, was accompanied with DeltaEtk:ER activation. Moreover, the conditionally activated Etk stimulated STAT1- and STAT5-dependent reporter activities by approximately 160- and approximately 15-fold, respectively, however, elicited only a modest STAT3-mediated reporter activation. Qualitatively comparable results were obtained in lung A549 cells, indicating that DeltaEtk:ER inducible system could function in an analogous fashion in different epithelial cells. Furthermore, we demonstrated that Etk activation alone augmented cyclin D1 promoter/enhancer activity via its STAT5 response element in both Pa-4DeltaEtk:ER and A549 cells. Altogether, these findings support the notion that the activation of Etk kinase is sufficient to transactivate STAT-mediated gene expression. Hence, our inducible DeltaEtk:ER system represents a novel approach to investigate the biochemical events following Etk activation and to evaluate the contribution by kinase activation of Etk alone or in conjunction with other signaling pathway(s) to the ultimate biological responses.

  12. Clonal evolution of AML on novel FMS-like tyrosine kinase-3 (FLT3 inhibitor therapy with evolving actionable targets

    Directory of Open Access Journals (Sweden)

    Pashtoon M. Kasi

    2016-01-01

    Full Text Available For acute myeloid leukemia (AML, identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3 has led to the development of several FLT3-inhibitors. Here we present clinical and next generation sequencing data at the time of progression of a patient on a novel FLT3-inhibitor clinical trial (ASP2215 to show that employing therapeutic interventions with these novel targeted therapies can lead to consequences secondary to selective pressure and clonal evolution of cancer. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process. (Clinical Trial: NCT02014558; registered at: 〈https://clinicaltrials.gov/ct2/show/NCT02014558〉

  13. 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.

  14. Inibidores de tirosino quinase na leucemia mieloide crônica Tyrosine kinase inhibitors in chronic myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Nei R. Lopes

    2009-01-01

    Full Text Available A leucemia mieloide crônica (LMC é uma neoplasia da medula óssea originada da translocação entre os cromossomos 9 e 22 t(9:22(q34;11 e forma o gene híbrido BCR-ABL, que possui intensa atividade tirosino quinase, sendo responsável pela proliferação das células tumorais. Um grande avanço no tratamento da LMC foi conquistado com o surgimento dos inibidores da tirosino quinase, entre eles o imatinibe, que vem demonstrando ser efetivo na maior parte dos pacientes com LMC por apresentar respostas duradouras. Entretanto, há pacientes resistentes ou que desenvolvem resistência durante o tratamento com esta droga; sendo assim, inibidores de tirosino quinase de segunda geração, como o dasatinibe e o nilotinibe, foram desenvolvidos apresentando maior potência com a finalidade de diminuir a chance de desenvolvimento de resistência. O bosutinibe e o INNO-406 estão sendo estudados para atender pacientes resistentes às drogas anteriormente citadas e também com a finalidade de diminuir efeitos colaterais das mesmas; entretanto, eles ainda estão em fase clínica de estudo. Há ainda outras drogas inibidoras da tirosino quinase que estão em desenvolvimento na fase clínica ou pré-clínica. A partir do desenvolvimento destas novas drogas, múltiplas opções de tratamento para os pacientes com LMC poderão ser propostas, podendo, desta forma, individualizar o tratamento de acordo com o que cada paciente necessita. Este estudo visa descrever as drogas antineoplásicas que têm como mecanismo de ação a inibição da enzima tirosino quinase na LMC.Chronic myeloid leukemia (CML is a neoplastic transformation of the hematopoietic system resulting from a t(9;22(q34;q11 translocation forming a BCR-ABL hybrid gene which has intense enzyme tyrosine kinase activity responsible for the proliferation of tumor cells. A dramatic positive response was achieved in CML patients with imatinib. This drug is effective in most patients because it presents long

  15. Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target.

    Science.gov (United States)

    Chiba, Shuntaro; Ikeda, Kazuyoshi; Ishida, Takashi; Gromiha, M Michael; Taguchi, Y-H; Iwadate, Mitsuo; Umeyama, Hideaki; Hsin, Kun-Yi; Kitano, Hiroaki; Yamamoto, Kazuki; Sugaya, Nobuyoshi; Kato, Koya; Okuno, Tatsuya; Chikenji, George; Mochizuki, Masahiro; Yasuo, Nobuaki; Yoshino, Ryunosuke; Yanagisawa, Keisuke; Ban, Tomohiro; Teramoto, Reiji; Ramakrishnan, Chandrasekaran; Thangakani, A Mary; Velmurugan, D; Prathipati, Philip; Ito, Junichi; Tsuchiya, Yuko; Mizuguchi, Kenji; Honma, Teruki; Hirokawa, Takatsugu; Akiyama, Yutaka; Sekijima, Masakazu

    2015-11-26

    A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective.

  16. Dietary phytoestrogens present in soy dramatically increase cardiotoxicity in male mice receiving a chemotherapeutic tyrosine kinase inhibitor.

    Science.gov (United States)

    Harvey, Pamela Ann; Leinwand, Leslie Anne

    2015-01-05

    Use of soy supplements to inhibit cancer cell growth is increasing among patients due to the perception that phytoestrogens in soy inhibit carcinogenesis via induction of apoptosis. Genistein, the most prevalent phytoestrogen in soy, is a potent endocrine disruptor and tyrosine kinase inhibitor (TKI) that causes apoptosis in many cells types. Chemotherapeutic TKIs limit cancer cell growth via the same mechanisms. However, TKIs such as Sunitinib cause cardiotoxicity in a significant number of patients. Molecular interactions between Sunitinib and dietary TKIs like genistein have not been examined in cardiomyocytes. Significant lethality occurred in mice treated with Sunitinib and fed a phytoestrogen-supplemented diet. Isolated cardiomyocytes co-treated with genistein and Sunitinib exhibited additive inhibition of signaling molecules important for normal cardiac function and increased apoptosis compared with Sunitinib alone. Thus, dietary soy supplementation should be avoided during administration of Sunitinib due to exacerbated cardiotoxicity, despite evidence for positive effects in cancer.

  17. A novel Bruton's tyrosine kinase gene (BTK) invariant splice site mutation in a Malaysian family with X-linked agammaglobulinemia.

    Science.gov (United States)

    Chear, Chai Teng; Gill, Harvindar Kaur; Ramly, Nazatul Haslina; Dhaliwal, Jasbir Singh; Bujang, Noraini; Ripen, Adiratna Mat; Mohamad, Saharuddin Bin

    2013-12-01

    X-linked agammaglobulinemia (XLA) is a rare genetic disorder caused by mutations in the Bruton's tyrosine kinase (BTK) gene. These mutations cause defects in early B cell development. A patient with no circulating B cells and low serum immunoglobulin isotypes was studied as were his mother and sister. Monocyte BTK protein expression was evaluated by flow cytometry. The mutation was determined using PCR and followed by sequencing. Flow cytometry showed the patient lacked BTK protein expression in his monocytes while the mother and sister had 62% and 40% of the monocytes showing BTK protein expressions respectively. The patient had a novel base substitution in the first nucleotide of intron 9 in the BTK gene, and the mutation was IVS9+1Gagammaglobulinemia and may be used for subsequent genetic counseling, carrier detection and prenatal diagnosis.

  18. Nonreceptor tyrosine kinase BMX maintains self-renewal and tumorigenic potential of glioblastoma stem cells by activating STAT3.

    Science.gov (United States)

    Guryanova, Olga A; Wu, Qiulian; Cheng, Lin; Lathia, Justin D; Huang, Zhi; Yang, Jinbo; MacSwords, Jennifer; Eyler, Christine E; McLendon, Roger E; Heddleston, John M; Shou, Weinian; Hambardzumyan, Dolores; Lee, Jeongwu; Hjelmeland, Anita B; Sloan, Andrew E; Bredel, Markus; Stark, George R; Rich, Jeremy N; Bao, Shideng

    2011-04-12

    Glioblastomas display cellular hierarchies containing tumor-propagating glioblastoma stem cells (GSCs). STAT3 is a critical signaling node in GSC maintenance but molecular mechanisms underlying STAT3 activation in GSCs are poorly defined. Here we demonstrate that the bone marrow X-linked (BMX) nonreceptor tyrosine kinase activates STAT3 signaling to maintain self-renewal and tumorigenic potential of GSCs. BMX is differentially expressed in GSCs relative to nonstem cancer cells and neural progenitors. BMX knockdown potently inhibited STAT3 activation, expression of GSC transcription factors, and growth of GSC-derived intracranial tumors. Constitutively active STAT3 rescued the effects of BMX downregulation, supporting that BMX signals through STAT3 in GSCs. These data demonstrate that BMX represents a GSC therapeutic target and reinforces the importance of STAT3 signaling in stem-like cancer phenotypes.

  19. Melanoma-associated antigen expression and the efficacy of tyrosine kinase inhibitors in head and neck cancer

    DEFF Research Database (Denmark)

    Hartmann, Stefan; Brands, Roman C; Küchler, Nora;

    2015-01-01

    receptor (EGFR). The efficacy of tyrosine kinase inhibitors (TKI) in the context of melanoma-associated antigens is discussed in the present study. Five human squamous cell carcinoma cell lines were treated with the EGFR TKIs, erlotinib and gefitinib. The efficacy of these agents was measured using......Melanoma-associated antigen (MAGE) has been identified in a variety of types of cancer. The expression of several MAGE subgroups is correlated with poor prognosis and chemotherapeutic resistance. One target of chemotherapeutic treatment in head and neck cancer is the epidermal growth factor...... correlation between MAGE-A5 and -A11 and lower efficacy of EGFR TKIs. Pretreatment analysis of MAGE-A status may therefore aid improvement of chemoprevention using erlotinib and gefitinib in head and neck cancer....

  20. Repositioning of Tyrosine Kinase Inhibitors as Antagonists of ATP-Binding Cassette Transporters in Anticancer Drug Resistance

    Directory of Open Access Journals (Sweden)

    Yi-Jun Wang

    2014-09-01

    Full Text Available The phenomenon of multidrug resistance (MDR has attenuated the efficacy of anticancer drugs and the possibility of successful cancer chemotherapy. ATP-binding cassette (ABC transporters play an essential role in mediating MDR in cancer cells by increasing efflux of drugs from cancer cells, hence reducing the intracellular accumulation of chemotherapeutic drugs. Interestingly, small-molecule tyrosine kinase inhibitors (TKIs, such as AST1306, lapatinib, linsitinib, masitinib, motesanib, nilotinib, telatinib and WHI-P154, have been found to have the capability to overcome anticancer drug resistance by inhibiting ABC transporters in recent years. This review will focus on some of the latest and clinical developments with ABC transporters, TKIs and anticancer drug resistance.

  1. Case of soluble fms-like tyrosine kinase 1 apheresis in severe pre-eclampsia developed at 15 weeks' gestation.

    Science.gov (United States)

    Nakakita, Baku; Mogami, Haruta; Kondoh, Eiji; Tsukamoto, Tatsuo; Yanagita, Motoko; Konishi, Ikuo

    2015-10-01

    Soluble fms-like tyrosine kinase-1 (sFlt1), a circulating vascular endothelial growth factor receptor 1 antagonist, is associated with the pathogenesis of pre-eclampsia. Extracorporeal removal of sFlt1 (sFlt1 apheresis) is emerging as a treatment for pre-eclampsia. We performed sFlt1 apheresis for a patient with very early onset pre-eclampsia, beginning at 15 weeks' gestation. She underwent sFlt1 apheresis 13 times from 19 to 23 weeks' gestation. The series of treatments lowered circulating sFlt1, stabilized blood pressure, reduced urinary protein, and preserved renal function, which contributed to a successful prolongation of pregnancy for 4 weeks and a live birth at 23(+3) weeks' gestation. Further studies are necessary for clinical application of sFlt1 apheresis as sFlt1 might have a protective function for the placenta and fetus in pre-eclampsia.

  2. [Tyrosine kinase inhibiting the VEGF pathway and elderly people: Tolerance, pre-treatment assessment and side effects management].

    Science.gov (United States)

    Bretagne, Marie; Boudou-Rouquette, Pascaline; Huillard, Olivier; Thomas-Schoemann, Audrey; Chahwakilian, Anne; Orvoen, Galdric; Arrondeau, Jennifer; Tlemsani, Camille; Cessot, Anatole; Cabanes, Laure; Blanchet, Benoit; Coriat, Romain; Alexandre, Jérôme; Goldwasser, François

    2016-03-01

    Angiogenesis inhibition is a major antitumor strategy that has emerged during the last decade. Oral tyrosine kinase inhibitors (TKI) targeting the VEGF receptor, including sunitinib, sorafenib, axitinib, regorafenib, pazopanib, and vandetanib reduce tumor growth and metastasis. These agents are approved for the treatment of metastatic diseases in first or second-line. They display a narrow therapeutic index. However, data in the elderly and/or in patients with multiple illnesses remain scarce. This population is classically excluded from clinical trials. The aim of this review is to provide an overview of existing literature regarding antiangiogenic TKI tolerance in the elderly (>70 years old). We also highlight key points of the pre-therapeutic evaluation and summarize the management of common toxicities.

  3. Deorphanization of the human leukocyte tyrosine kinase (LTK) receptor by a signaling screen of the extracellular proteome

    Science.gov (United States)

    Zhang, Hongbing; Pao, Lily I.; Zhou, Aileen; Brace, Arthur D.; Halenbeck, Robert; Hsu, Amy W.; Bray, Thomas L.; Hestir, Kevin; Bosch, Elizabeth; Lee, Ernestine; Wang, Gang; Liu, Haixia; Wong, Brian R.; Kavanaugh, W. Michael; Williams, Lewis T.

    2014-01-01

    There are many transmembrane receptor-like proteins whose ligands have not been identified. A strategy for finding ligands when little is known about their tissue source is to screen each extracellular protein individually expressed in an array format by using a sensitive functional readout. Taking this approach, we have screened a large collection (3,191 proteins) of extracellular proteins for their ability to activate signaling of an orphan receptor, leukocyte tyrosine kinase (LTK). Only two related secreted factors, FAM150A and FAM150B (family with sequence similarity 150 member A and member B), stimulated LTK phosphorylation. FAM150A binds LTK extracellular domain with high affinity (KD = 28 pM). FAM150A stimulates LTK phosphorylation in a ligand-dependent manner. This strategy provides an efficient approach for identifying functional ligands for other orphan receptors. PMID:25331893

  4. Antidepressant drug exposure is associated with mRNA levels of tyrosine receptor kinase B in major depressive disorder.

    Science.gov (United States)

    Bayer, T A; Schramm, M; Feldmann, N; Knable, M B; Falkai, P

    2000-08-01

    1. Recent studies have provided support for the notion that the high affinity neurotrophin receptor tyrosine receptor kinase B (trk B) may be involved in the treatment of depression. 2. Using a quantitative RT-PCR approach trk B mRNA levels were determined in brain material from cerebellum, temporal cortex, and frontal cortex of control specimen and patients with major depressive disorder, schizophrenia and bipolar disorder (15 subjects each). 3. Interestingly, elevated trk B mRNA levels were found in cerebellum (3.6-fold) in patients with major depressive disorder, reaching statistical significance (p=0.03). 4. The major depressive disorder-on drugs group differed from controls (p=0.006) in the cerebellum. 5. Since only patients with major depressive disorder received antidepressants, elevated trk B mRNA levels are possibly related to drug treatment.

  5. [Role of Bone Marrow Mesenchymal Stem Cells in Resistance of Chronic Myeloid Leukemia to Tyrosine Kinase Inhibitors -Review].

    Science.gov (United States)

    Zhang, Xiao-Yan; Wan, Qian; Fang, Li-Jun; Li, Jian

    2016-12-01

    Chronic myeloid leukemia (CML) is a disease originated from malignant hematopoietic stem cell disorder. In CML, mesenchymal stem cells(MSC) have been changed in the bone marrow microenvironment, which can protect the leukemia cells from apoptosis induced by tyrosine kinase inhibitors (TKI) and lead to the resistance to TKI by the secretion of soluble factors, involvement in cell-cell adhesion, and so on. This review mainly focuses on the changes of the bone marrow mesenchymal stem cells in CML, as well as the role and mechanism of MSC in the CML resistance of TKI. The concrete probrems dicussing in this review are role of MSC in bone marrow microenviroment, characteristics of MSC in CML, the related mechanisms of MSC in drug resistance and so on.

  6. RTK SLAP down: the emerging role of Src-like adaptor protein as a key player in receptor tyrosine kinase signaling.

    Science.gov (United States)

    Wybenga-Groot, Leanne E; McGlade, C Jane

    2015-02-01

    SLAP (Src like adaptor protein) contains adjacent Src homology 3 (SH3) and Src homology 2 (SH2) domains closely related in sequence to that of cytoplasmic Src family tyrosine kinases. Expressed most abundantly in the immune system, SLAP function has been predominantly studied in the context of lymphocyte signaling, where it functions in the Cbl dependent downregulation of antigen receptor signaling. However, accumulating evidence suggests that SLAP plays a role in the regulation of a broad range of membrane receptors including members of the receptor tyrosine kinase (RTK) family. In this review we highlight the role of SLAP in the ubiquitin dependent regulation of type III RTKs PDGFR, CSF-1R, KIT and Flt3, as well as Eph family RTKs. SLAP appears to bind activated type III and Eph RTKs via a conserved autophosphorylated juxtamembrane tyrosine motif in an SH2-dependent manner, suggesting that SLAP is important in regulating RTK signaling.

  7. Cellular maturation defects in Bruton's tyrosine kinase-deficient immature B cells are amplified by premature B cell receptor expression and reduced by receptor editing

    NARCIS (Netherlands)

    S. Middendorp; R.W. Hendriks (Rudi)

    2004-01-01

    textabstractIn the mouse, Bruton's tyrosine kinase (Btk) is essential for efficient developmental progression of CD43(+)CD2(-) large cycling into CD43(-)CD2(+) small resting pre-B cells in the bone marrow and of IgM(high) transitional type 2 B cells into IgM(low) mature B cells in

  8. Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase.

    NARCIS (Netherlands)

    G.M. Dingjan (Gemma); A. Maas (Alex); M.C. Nawijn (Martijn); L. Smit (Linda); J.S. Voerman (Jane); R.W. Hendriks (Rudi); F.G. Grosveld (Frank)

    1998-01-01

    textabstractTo identify B-cell signaling pathways activated by Bruton's tyrosine kinase (Btk) in vivo, we generated transgenic mice in which Btk expression is driven by the MHC class II Ea gene locus control region. Btk overexpression did not have significant adverse ef

  9. Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase

    NARCIS (Netherlands)

    Dingjan, GM; Maas, A; Nawijn, MC; Smit, L; Voerman, JSA; Grosveld, F; Hendriks, RW

    1998-01-01

    To identify B-cell signaling pathways activated by Bruton's tyrosine kinase (Btk) in vivo, we generated transgenic mice in which Btk expression is driven by the MHC class II Ea gene locus control region. Btk overexpression did not have significant adverse effects on B cell function, and essentially

  10. Bruton's tyrosine kinase gene mutations in Turkish patients with X-linked agammaglobulinemia from a single center: Novel mutations in βTK gene

    NARCIS (Netherlands)

    Ç. Aydoǧmuş (Çiǧdem); Y. Camcioǧlu (Yildiz); M. van der Burg (Mirjam); H. Çokuǧraş (H.); N. Akçakaya (Necla); J.J.M. van Dongen (Jacques)

    2013-01-01

    textabstractObjective: X-linked agammaglobulinemia (XLA) is caused by a mutation in the Bruton's tyrosine kinase gene and is characterized by a delay in the maturation and differentiation of B lymphocytes. Patients with XLA have either absent or very low levels of circulating mature B cells (<1%), p

  11. Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction

    NARCIS (Netherlands)

    Sparidans, Rolf W; van Hoppe, Stephanie; Rood, Johannes J M; Schinkel, Alfred H; Schellens, Jan H M; Beijnen, Jos H

    2016-01-01

    A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out as

  12. Potent quinoxaline-based inhibitors of PDGF receptor tyrosine kinase activity. Part 2: the synthesis and biological activities of RPR127963 an orally bioavailable inhibitor.

    Science.gov (United States)

    He, Wei; Myers, Michael R; Hanney, Barbara; Spada, Alfred P; Bilder, Glenda; Galzcinski, Helen; Amin, Dilip; Needle, Saul; Page, Ken; Jayyosi, Zaid; Perrone, Mark H

    2003-09-15

    RPR127963 demonstrates an excellent pharmacokinetic profile in several species and was found to be efficacious in the prevention of restenosis in a Yucatan mini-pig model upon oral administration of 1-5 mg/kg. The in vitro selectivity profile and SAR of the highly optimized PDGF-R tyrosine kinase inhibitor are highlighted.

  13. Serum concentrations of nitrite and malondialdehyde as markers of oxidative stress in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

    Directory of Open Access Journals (Sweden)

    Maria Juracy Petrola

    2012-01-01

    Full Text Available BACKGROUND: Chronic myeloid leukemia is a neoplasm characterized by clonal expansion of hematopoietic progenitor cells resulting from the (9:22(q34,11 translocation. The tyrosine kinase abl fusion protein,the initial leukemogenic event in chronic myeloid leukemia, is constitutively activated thus inducing the production of reactive oxygen species. Of particular relevance is the fact that an increase in reactive oxygen species can facilitate genomic instability and may contribute to disease progression. OBJETIVE: To evaluate oxidative stress by determining the levels of malondialdehyde and nitrite in chronic myeloid leukemia patients under treatment with 1st and 2nd generation tyrosine kinase inhibitors monitored at a referral hospital in Fortaleza, Ceará. METHODS: A cross-sectional study was performed of 64 male and female adults. Patients were stratified according to treatment. The levels of malondialdehyde and nitrite were determined by spectrophotometry. Statistical differences between groups were observed using the Student t-test and Fisher's exact test. The results are expressed as mean ± standard error of mean. The significance level was set for a p-value < 0.05 in all analyses. RESULTS: The results show significantly higher mean concentrations of nitrite and malondialdehyde in chronic myeloid leukemia patients using second-generation tyrosine kinase inhibitors compared to patients on imatinib. Conclusion: It follows that chronic myeloid leukemia patients present higher oxidative activity and that the increases in oxidative damage markers can indicate resistance to 1st generation tyrosine kinase inhibitors.

  14. Effect of tyrosine kinase inhibitor treatment of renal cell carcinoma on the accumulation of carbonic anhydrase IX-specific chimeric monoclonal antibody cG250

    NARCIS (Netherlands)

    Oosterwijk-Wakka, J.C.; Kats-Ugurlu, G.; Leenders, W.P.J.; Kiemeney, L.A.L.M.; Old, L.J.; Mulders, P.F.A.; Oosterwijk, E.

    2011-01-01

    OBJECTIVE: To investigate the effect of three different tyrosine kinase inhibitors (TKIs) on the biodistribution of chimeric monoclonal antibody (mAb) cG250, which identifies carbonic anhydrase IX (CAIX), in nude mice bearing human renal cell carcinoma (RCC) xenografts. TKIs represent the best, but

  15. Combined treatment of tyrosine kinase inhibitor labeled gold nanorod encapsulated albumin with laser thermal ablation in a renal cell carcinoma model

    Science.gov (United States)

    This manuscript served to characterize and evaluate Human Serum Albumin-encapsulated Nanoparticles (NPs) for drug delivery of a tyrosine kinase inhibitor combined with induction of photothermal ablation (PTA) combination therapy of Renal Cell Carcinoma (RCC). RCC is the most common type of kidney c...

  16. Increased serum levels of anti-angiogenic factors soluble fms-like tyrosine kinase and soluble endoglin in sickle cell disease

    NARCIS (Netherlands)

    Landburg, P.P.; Elsenga, H.; Schnog, J.B.; Duits, A.J.

    2008-01-01

    The anti-angiogenic factors soluble fms-like tyrosine kinase (sFlt)-1 and soluble endoglin (sEng) have been shown to be of importance in angiogenesis by sequestering and inhibiting vascular endothelial growth factor, placenta-like growth factor and transforming growth factor-beta(1) signaling. Given

  17. The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats

    DEFF Research Database (Denmark)

    Sarin, C T; Tack, B F; Kristensen, Torsten;

    1986-01-01

    We have isolated and sequenced a full-length cDNA clone for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain). This sequence predicts a 339 amino acid (Mr 38,493) protein containing an N-terminal region of 20 amino acids, known to interact with a 10 kd protein (light chain), and...

  18. [Literature review and presentation of our own research results regarding the effects on bone of tyrosine kinase inhibitors imatinib and nilotinib used in the treatment of oncohematological diseases].

    Science.gov (United States)

    Kirschner, Gyöngyi; Balla, Bernadett; Kósa, János; Horváth, Péter; Kövesdi, Andrea; Lakatos, Gergely; Takács, István; Nagy, Zsolt; Tóbiás, Bálint; Árvai, Kristóf; Lakatos, Péter

    2016-09-01

    Tyrosine kinase inhibitors are widely used for treatment of certain oncohematological diseases. Several clinical studies have confirmed that specific BCR-ABL tyrosine kinase inhibitors alter the physiological process of bone tissue in a complex and unclearly identified manner. Since these treatments are being given to more and more patients, and the therapy takes decades or lasts even lifelong, it is justifiable to obtain more detailed knowledge of the molecular background of these mechanisms. In this article the authors summarize preliminary research results and human clinical observations on imatinib and nilotinib which are related to bone metabolism, and present the results of their own experiments in in vitro osteoblast cultures. Based on the presented results, the effects of imatinib and nilotinib on bone cells depend on the concentration of imatinib and nilotinib, the maturation stage of the cells and the distribution ratio of receptor tyrosine kinase signaling pathways. In this study the authors firstly prepared a stop-gap, comprehensive review in the Hungarian literature, regarding the effects of tyrosine kinase inhibitors on bone metabolism. In addition they firstly performed whole transcriptome analysis on osteoblasts in order to obtain a better understanding of the cellular molecular mechanisms. Orv. Hetil., 2016, 157(36), 1429-1437.

  19. Combination therapy with gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, gemcitabine and cisplatin in patients with advanced solid tumors.

    NARCIS (Netherlands)

    Giaccone, G.; Gonzalez-Larriba, JL; Oosterom, van A.T.; Alfonso, R; Smit, E.F.; Martens, M.; Peters, G.J.; Vijgh, van der WJ; Smith, R; Averbuch, S; Fandi, A

    2004-01-01

    BACKGROUND: The aim of this study was to investigate the tolerability, pharmacokinetic interaction and antitumor activity of gefitinib ("Iressa", ZD1839), an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, combined with gemcitabine and cisplatin in chemotherapy-n

  20. Src-Like adaptor protein (SLAP) binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

    Science.gov (United States)

    Kazi, Julhash U; Rönnstrand, Lars

    2012-01-01

    Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  1. Src-Like adaptor protein (SLAP binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

    Directory of Open Access Journals (Sweden)

    Julhash U Kazi

    Full Text Available Fms-like tyrosine kinase 3 (Flt3 is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML. Src-like adaptor protein (SLAP is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  2. EGFR tyrosine kinase inhibitory peptide attenuates Helicobacter pylori-mediated hyper-proliferation in AGS enteric epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Himaya, S.W.A. [Marine Bio-Process Research Center, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Dewapriya, Pradeep [Department of Chemistry, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Kim, Se-Kwon, E-mail: sknkim@pknu.ac.kr [Marine Bio-Process Research Center, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Department of Chemistry, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of)

    2013-06-15

    Helicobacter pylori infection is one of the most critical causes of stomach cancer. The current study was conducted to explore the protective effects of an isolated active peptide H-P-6 (Pro-Gln-Pro-Lys-Val-Leu-Asp-Ser) from microbial hydrolysates of Chlamydomonas sp. against H. pylori-induced carcinogenesis. The peptide H-P-6 has effectively suppressed H. pylori-induced hyper-proliferation and migration of gastric epithelial cells (AGS). However, the peptide did not inhibit the viability of the bacteria or invasion into AGS cells. Therefore, the effect of the peptide on regulating H. pylori-induced molecular signaling was investigated. The results indicated that H. pylori activates the EGFR tyrosine kinase signaling and nuclear translocation of the β-catenin. The EGFR activation has led to the up-regulation of PI3K/Akt signaling pathway. Moreover, the nuclear translocation levels of β-catenin were significantly increased as a result of Akt mediated down-regulation of GSK3/β protein levels in the cytoplasm. Both of these consequences have resulted in increased expression of cell survival and migration related genes such as c-Myc, cyclin-D, MMP-2 and matrilysin. Interestingly, the isolated peptide potently inhibited H. pylori-mediated EGFR activation and thereby down-regulated the subsequent P13K/Akt signaling leading to β-catenin nuclear translocation. The effect of the peptide was confirmed with the use of EGFR tyrosine kinase inhibitor AG1487 and molecular docking studies. Collectively this study identifies a potent peptide which regulates the H. pylori-induced hyper-proliferation and migration of AGS cells at molecular level. - Highlights: • Chlamydomonas sp. derived peptide H-P-6 inhibits H. pylori-induced pathogenesis. • H-P-6 suppresses H. pylori-induced hyper-proliferation and migration of AGS cells. • The peptide inhibits H. pylori-induced EGFR activation.

  3. Three generations of epidermal growth factor receptor tyrosine kinase inhibitors developed to revolutionize the therapy of lung cancer

    Directory of Open Access Journals (Sweden)

    Zhang H

    2016-11-01

    Full Text Available Haijun Zhang Department of Oncology, Zhongda Hospital, Medical School, Southeast University, Nanjing, People’s Republic of China Abstract: Lung cancer, ~80%–85% of which is non-small-cell lung cancer (NSCLC, is the leading cause of cancer-related mortality worldwide. Sensitizing mutations in epidermal growth factor receptor (EGFR gene (EGFRm+, such as exon 19 deletions and exon 21 L858R point mutations, are the most important drivers in NSCLC patients. In this respect, small-molecule EGFR tyrosine kinase inhibitors (TKIs have been designed and developed, which launched the era of targeted, personalized and precise medicine for lung cancer. Patients with EGFRm+ could achieve good responses to the treatment with the first-generation EGFR TKIs, such as erlotinib and gefitinib. However, most patients develop acquired drug resistance mostly driven by the T790M mutation occurring within exon 20. Although the second-generation EGFR TKIs, such as afatinib, dacomitinib and neratinib, demonstrated promising activity against T790M in preclinical models, they have failed to overcome resistance in patients due to dose-limiting toxicity. Recently, the third-generation EGFR TKIs have shown to be effective against cell lines and murine models harboring T790M mutations while sparing wild-type EGFR, which represents a promising breakthrough approach in overcoming T790M-mediated resistance in NSCLC patients. This article provides a comprehensive review of the therapy revolution for NSCLC with three generations of EGFR TKIs. Keywords: lung cancer, epidermal growth factor receptor, tyrosine kinase inhibitors, T790M mutation

  4. Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.

    Directory of Open Access Journals (Sweden)

    Naadiya Carrim

    Full Text Available We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.Human and mouse washed platelets (from WT or Pyk2 KO mice were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P surface expression and integrin αIIbβ3 activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk, PI3-K and Bruton's tyrosine kinase (Btk and upstream of Rac1, PLCγ2, Ca2+ release, PKC, Hic-5, NOX1 and αIIbβ3 activation.Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.

  5. Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors.

    Science.gov (United States)

    Davare, Monika A; Vellore, Nadeem A; Wagner, Jacob P; Eide, Christopher A; Goodman, James R; Drilon, Alexander; Deininger, Michael W; O'Hare, Thomas; Druker, Brian J

    2015-09-29

    Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib's dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1(G2032R) mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure-function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies.

  6. "Screening of the Bruton Tyrosine Kinase (BTK Gene Mutations in 13 Iranian Patients with Presumed X-Linked Agammaglobulinemia "

    Directory of Open Access Journals (Sweden)

    "Asghar Aghamohammadi

    2004-12-01

    Full Text Available X-linked agammaglobulinemia (XLA is an immunodeficiency caused by mutations in the Bruton tyrosine kinase (Btk gene. In order to identify the mutations in Btk gene in Iranian patients with antibody deficiency, 13 male patients with an XLA phenotype from 11 unrelated families were enrolled as the subjects of investigation for Btk mutation analysis using PCR-SSCP followed by sequencing. Five different mutations were identified in 5 patients from 5 unrelated families. Three mutations had been reported previously including TTTG deletion in intron 15 (4 bps upstream of exon 16 boundary, nonsense point mutation (1896G>A that resulted in a premature stop codon (W588X in kinase domain, and nucleotide alteration in invariant splice donor site of exon12 (IVS12+1G>A. While 2 novel missense mutations (2084A>G, 1783T>C were identified leading to amino acid changes (I651T, Y551H. The results of this study further support the notion that molecular genetic testing represents an important tool for definitive and early diagnosis of XLA and may allow accurate carrier detection and prenatal diagnosis.

  7. The glypiated neuronal cell adhesion molecule contactin/F11 complexes with src-family protein tyrosine kinase Fyn.

    Science.gov (United States)

    Zisch, A H; D'Alessandri, L; Amrein, K; Ranscht, B; Winterhalter, K H; Vaughan, L

    1995-06-01

    Glycosyl phosphatidylinositol-anchored glycoproteins of the immunoglobulin superfamily play an important role in the formation of neuronal networks during development. The mechanism whereby neuronal GPI-linked molecules transduce recognition signals remains to be established. Analysis of detergent-resistant immune-complexes reveals that the glypiated neuronal cell adhesion molecule contactin/F11 specifically complexes with the cytoplasmic, nonreceptor type src-family tyrosine kinase Fyn. Antibody-mediated cross-linking of contactin/F11 on embryonic chick neuronal cells leads to an increase of the Fyn-activity coprecipitated with contactin/F11, and elevates phosphorylation of an additional 75/80 K component within the contactin/F11-immune-complex. Additionally, binding of ligands, i.e., contactin/F11-specific antibody or tenascin-R, a natural ligand of contactin/F11, to the surface of HeLa transfectants expressing contactin/F11, causes capping of contactin/F11 and a concomitant change in the distribution of the intracellular kinase Fyn, thus confirming their physical association. This indicates that contactin/F11-mediated signaling requires Fyn.

  8. Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

    LENUS (Irish Health Repository)

    Manser, C

    2012-05-31

    A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3β (GSK3β). KLC2 phosphorylation by GSK3β induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-β (TGFβ) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFβ-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

  9. MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells.

    Science.gov (United States)

    Mukherjee, P; Tinder, T L; Basu, G D; Gendler, S J

    2005-01-01

    MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex.

  10. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases.

    Science.gov (United States)

    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J; Nie, Guangjun

    2016-04-15

    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment.

  11. The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel.

    Science.gov (United States)

    Fischer, H; Machen, T E

    1996-12-01

    The role of the tyrosine kinase p60c-src on the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel was investigated with the cell-attached and excised patch clamp technique in conjunction with current noise analysis of recordings containing multiple channels per patch. Spectra of CFTR-generated current noise contained a low-frequency and a high-frequency Lorentzian noise component. In the cell-attached mode, the high-frequency Lorentzian was significantly dependent on the membrane potential, while the low-frequency Lorentzian was unaffected. Excision of forskolin-stimulated patches into ATP-containing solution significantly reduced the amplitude of the voltage-dependent high-frequency Lorentzian. Addition of the tyrosine kinase p60c-src to excised, active, CFTR-containing membrane patches increased mean currents by 54%, increased the corner frequency of the low-frequency Lorentzian, and recovered the high-frequency Lorentzian and its characteristics. Treatment with lambda-phosphatase inactivated src-induced currents and changes in gating. When active patches were excised under conditions in which patch-associated tyrosine phosphatases were blocked with sodium vanadate, the high-frequency gating remained relatively unchanged. The results suggest that CFTR's open probability and its voltage-dependent fast gate are dependent on tyrosine phosphorylation, and that membrane-associated tyrosine phosphatases are responsible for inactivation of the fast gate after patch excision.

  12. STAT3 activation in pressure-overloaded feline myocardium: role for integrins and the tyrosine kinase BMX.

    Science.gov (United States)

    Willey, Christopher D; Palanisamy, Arun P; Johnston, Rebecca K; Mani, Santhosh K; Shiraishi, Hirokazu; Tuxworth, William J; Zile, Michael R; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

    2008-06-27

    Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypertrophy. Signal transducer and activator of transcription-3 (STAT3) activation is an important cardioprotective factor associated with cardiac hypertrophy. Although STAT3 activation has been reported via signaling through Janus Kinase 2 (JAK2) in several cardiac models of hypertrophy, the importance of other nonreceptor tyrosine kinases (NTKs) has not been explored. Utilizing an in vivo feline right ventricular pressure-overload (RVPO) model of hypertrophy, we demonstrate that in 48 h pressure-overload (PO) myocardium, STAT3 becomes phosphorylated and redistributed to detergent-insoluble fractions with no accompanying JAK2 activation. PO also caused increased levels of phosphorylated STAT3 in both cytoplasmic and nuclear fractions. To investigate the role of other NTKs, we used our established in vitro cell culture model of hypertrophy where adult feline cardiomyocytes are embedded three-dimensionally (3D) in type-I collagen and stimulated with an integrin binding peptide containing an Arg-Gly-Asp (RGD) motif that we have previously shown to recapitulate the focal adhesion complex (FAC) formation of 48 h RVPO. RGD stimulation of adult cardiomyocytes in vitro caused both STAT3 redistribution and activation that were accompanied by the activation and redistribution of c-Src and the TEC family kinase, BMX, but not JAK2. However, infection with dominant negative c-Src adenovirus was unable to block RGD-stimulated changes on either STAT3 or BMX. Further analysis in vivo in 48 h PO myocardium showed the presence of both STAT3 and BMX in the detergent-insoluble fraction with their complex formation and phosphorylation. Therefore, these studies indicate a novel mechanism of BMX-mediated STAT3 activation within a PO model of cardiac hypertrophy that might contribute to cardiomyocyte growth and survival.

  13. STAT3 Activation in Pressure-Overloaded Feline Myocardium: Role for Integrins and the Tyrosine Kinase BMX

    Directory of Open Access Journals (Sweden)

    Christopher D. Willey, Arun P. Palanisamy, Rebecca K. Johnston, Santhosh K. Mani, Hirokazu Shiraishi, William J. Tuxworth, Michael R. Zile, Sundaravadivel Balasubramanian, Dhandapani Kuppuswamy

    2008-01-01

    Full Text Available Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypertrophy. Signal transducer and activator of transcription-3 (STAT3 activation is an important cardioprotective factor associated with cardiac hypertrophy. Although STAT3 activation has been reported via signaling through Janus Kinase 2 (JAK2 in several cardiac models of hypertrophy, the importance of other nonreceptor tyrosine kinases (NTKs has not been explored. Utilizing an in vivo feline right ventricular pressure-overload (RVPO model of hypertrophy, we demonstrate that in 48 h pressure-overload (PO myocardium, STAT3 becomes phosphorylated and redistributed to detergent-insoluble fractions with no accompanying JAK2 activation. PO also caused increased levels of phosphorylated STAT3 in both cytoplasmic and nuclear fractions. To investigate the role of other NTKs, we used our established in vitro cell culture model of hypertrophy where adult feline cardiomyocytes are embedded three-dimensionally (3D in type-I collagen and stimulated with an integrin binding peptide containing an Arg-Gly-Asp (RGD motif that we have previously shown to recapitulate the focal adhesion complex (FAC formation of 48 h RVPO. RGD stimulation of adult cardiomyocytes in vitro caused both STAT3 redistribution and activation that were accompanied by the activation and redistribution of c-Src and the TEC family kinase, BMX, but not JAK2. However, infection with dominant negative c-Src adenovirus was unable to block RGD-stimulated changes on either STAT3 or BMX. Further analysis in vivo in 48 h PO myocardium showed the presence of both STAT3 and BMX in the detergent-insoluble fraction with their complex formation and phosphorylation. Therefore, these studies indicate a novel mechanism of BMX-mediated STAT3 activation within a PO model of cardiac hypertrophy that might contribute to cardiomyocyte growth and survival.

  14. A novel homolog of protein tyrosine kinase Fyn identified in Lampetra japonica with roles in the immune response.

    Science.gov (United States)

    Zhang, Qiong; Song, Xueying; Su, Peng; Li, Ranran; Liu, Chang; Gou, Meng; Wang, Hao; Liu, Xin; Li, Qingwei

    2016-04-01

    The non-receptor protein tyrosine kinase (nrPTK) Fyn, a member of the avian sarcoma virus transforming gene (Src) kinase family, plays a very significant role in cell growth, survival, apoptosis, tumor formation and immune response. In this study, a homolog of nrPTK Fyn was identified for the first time in the lamprey, Lampetra japonica and was named "Lja-Fyn". The cDNA fragment of lamprey lja-fyn contains a 1611-bp open reading frame, which encodes a protein of 537 amino acids. Multiple sequence alignment analysis showed that it shares four conserved domains (Src homology (SH) 4, SH3, SH2 and protein kinases catalytic domains) and a variable unique domain with vertebrates Fyn molecules. Though Lja-Fyn has high sequence similarity with typical Fyn and Yes molecules of jawed vertebrates, the identities among Lja-Fyn and typical Fyn molecules in unique domain are relatively higher than that among Lja-Fyn and typical Yes molecules. The result indicates that Lja-Fyn is a homolog of Fyn rather than Yes. The phylogenetic analysis showed that Fyn, Yes and Src molecules are grouped into three distinct phylogenetic clusters, and Lja-Fyn is grouped as a single branch in Fyn cluster. The real-time quantitative PCR assay revealed the wide distribution of the lja-fyn mRNA in lamprey immune related tissues. After stimulation with mixed antigens, the levels of lja-fyn mRNA were obviously up-regulated in the gill and lymphocyte-like cells, and the similar results were got by western blot analysis of Lja-Fyn protein expression. These results indicated that nrPTK Lja-Fyn was likely to be involved in immune response. Furthermore, our present findings also provide the necessary information for understanding the distinction between lamprey Lja-Fyn and other members of jawed vertebrates in Src family.

  15. Oncogenic tyrosine kinase NPM/ALK induces activation of the MEK/ERK signaling pathway independently of c-Raf.

    Science.gov (United States)

    Marzec, M; Kasprzycka, M; Liu, X; Raghunath, P N; Wlodarski, P; Wasik, M A

    2007-02-01

    The mechanisms of cell transformation mediated by the highly oncogenic, chimeric NPM/ALK tyrosine kinase remain only partially understood. Here we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma (ALK+ TCL) display phosphorylation of the extracellular signal-regulated protein kinase (ERK) 1/2 complex. Transfection of BaF3 cells with NPM/ALK induces phosphorylation of EKR1/2 and of its direct activator mitogen-induced extracellular kinase (MEK) 1/2. Depletion of NPM/ALK by small interfering RNA (siRNA) or its inhibition by WHI-154 abrogates the MEK1/2 and ERK1/2 phosphorylation. The NPM/ALK-induced MEK/ERK activation is independent of c-Raf as evidenced by the lack of MEK1/2 and ERK1/2 phosphorylation upon c-Raf inactivation by two different inhibitors, RI and ZM336372, and by its siRNA-mediated depletion. In contrast, ERK1/2 activation is strictly MEK1/2 dependent as shown by suppression of the ERK1/2 phosphorylation by the MEK1/2 inhibitor U0126. The U0126-mediated inhibition of ERK1/2 activation impaired proliferation and viability of the ALK+ TCL cells and expression of antiapoptotic factor Bcl-xL and cell cycle-promoting CDK4 and phospho-RB. Finally, siRNA-mediated depletion of both ERK1 and ERK2 inhibited cell proliferation, whereas depletion of ERK 1 (but not ERK2) markedly increased cell apoptosis. These findings identify MEK/ERK as a new signaling pathway activated by NPM/ALK and indicate that the pathway represents a novel therapeutic target in the ALK-induced malignancies.

  16. Tyrosine kinase of insulin-like growth factor receptor as target for novel treatment and prevention strategies of colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Michael H(o)pfner; Andreas P Sutter; Alexander Huether; Viola Baradari; Hans Scherübl

    2006-01-01

    AIM: To investigate the antineoplastic potency of the novel insulin-like growth factor 1 receptor (IGF-1R) tyrosine kinase inhibitor (TKI) NVP-AEW541 in cell lines and primary cell cultures of human colorectal cancer (CRC).METHODS: Cells of primary colorectal carcinomas were from 8 patients. Immunostaining and crystal violet staining were used for analysis of growth factor receptor protein expression and detection of cell number changes,respectively. Cytotoxicity was determined by measuring the release of the cytoplasmic enzyme lactate dehydrogenase (LDH). The proportion of apoptotic cells was determined by quantifying the percentage of sub-G1(hypodiploid) cells. Cell cycle status reflected by the DNA content of the nuclei was detected by flow cytometry.RESULTS: NVP-AEW541 dose-dependently inhibited the proliferation of colorectal carcinoma cell lines and primary cell cultures by inducing apoptosis and cell cycle arrest. Apoptosis was characterized by caspase-3 activation and nuclear degradation. Cell cycle was arrested at the G1/S checkpoint. The NVP-AEW541-mediated cell cycle-related signaling involved the inactivation of Akt and extracellular signal-regulated kinase (ERK) 1/2, the upregulation of the cyclin-dependent kinase inhibitors p21Waf1/CIP1 and p27Kip1, and the downregulation of the cell cycle promoter cyclin D1. Moreover, BAX was upregulated during NVP-AEW541-induced apoptosis, whereas Bcl-2 was downregulated. Measurement of LDH release showed that the antineoplastic effect of NVP-AEW541 was not due to general cytotoxicity of the compound.However, augmented antineoplastic effects were observed in combination treatments of NVP-AEW541 with either 5-FU, or the EGFR-antibody cetuximab, or the HMG-CoA-reductase inhibitor fluvastatin.CONCLUSION: IGF-1R-TK inhibition is a promising novel approach for either mono- or combination treatment strategies of colorectal carcinoma and even for CRC chemoprevention.

  17. Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

    Science.gov (United States)

    Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

    1997-06-05

    Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

  18. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro

    Science.gov (United States)

    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-01-01

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  19. Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor, is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons

    Directory of Open Access Journals (Sweden)

    Yuan Jian

    2010-06-01

    Full Text Available Abstract Background The downstream of tyrosine kinase/docking protein (Dok adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk receptor family, which has three members (TrkA, TrkB and TrkC, are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction. Results In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST precipitation assays and coimmunoprecipitation (Co-IP experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3 stimulation. Conclusions We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons.

  20. The Insect Neuropeptide PTTH Activates Receptor Tyrosine Kinase Torso to Initiate Metamorphosis

    DEFF Research Database (Denmark)

    Rewitz, Kim; Yamanaka, Naoki; Gilbert, Lawrence;

    2009-01-01

    Holometabolous insects undergo complete metamorphosis to become sexually mature adults. Metamorphosis is initiated by brain-derived prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone via an incompletely defined signaling pathway. Here we demonstrate...... in the prothoracic gland (PG), and its loss phenocopies the removal of PTTH. The activation of Torso by PTTH stimulates extracellular signal–regulated kinase (ERK) phosphorylation, and the loss of ERK in the PG phenocopies the loss of PTTH and Torso. We conclude that PTTH initiates metamorphosis by activation...

  1. Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase Inhibitors

    Science.gov (United States)

    2015-12-01

    transformation (11–14) (Figure 1). In normal cells, DSBs are repaired with high fidelity by members of the homologous recombination (HR) pathway which restore...kinase complement of the human genome. Science (New York, N.Y.), 298, 1912–1934. 34. Cosaceanu,D., Budiu,R.A., Carapancea,M., Castro ,J., Lewensohn,R...Interaction enhances end-joining fidelity of chromosomal doobll’-strand breaks In the G 1 phase of the cell cycle. J Bioi Chern 201 3; :ZII: 8966

  2. Dacomitinib in lung cancer: a “lost generation” EGFR tyrosine-kinase inhibitor from a bygone era?

    Directory of Open Access Journals (Sweden)

    Ou SH

    2015-10-01

    Full Text Available Sai-Hong Ignatius Ou,1 Ross A Soo21Chao Family Comprehensive Cancer Center, Division of Hematology/Oncology, Department of Medicine, University of California, Irvine School of Medicine, Orange, CA, USA; 2National University Health System and Cancer Science Institute of Singapore, SingaporeAbstract: EGFR tyrosine-kinase inhibitors (TKIs have now been firmly established as the first-line treatment for non-small-cell lung cancer (NSCLC patients harboring activating EGFR mutations, based on seven prospective randomized Phase III trials. However, despite significantly improved overall response rate and improved median progression-free survival when compared to platinum-doublet chemotherapy, EGFR-mutant NSCLC patients treated with EGFR TKIs invariably progress due to the emergence of acquired resistances, with the gatekeeper T790M mutation accounting for up to 60% of the resistance mechanisms. Second-generation irreversible EGFR TKIs were developed in part to inhibit the T790M mutation, in addition to the common activating EGFR mutations. Dacomitinib is one such second-generation EGFR TKI designed to inhibit both the wild-type (WT EGFR and EGFR T790M. Afatinib is another second-generation EGR TKI that has been now been approved for the first-line treatment of EGFR-mutant NSCLC patients, while dacomitinib continues to undergo clinical evaluation. We will review the clinical development of dacomitinib from Phase I to Phase III trials, including the two recently published negative large-scale randomized Phase III trials (ARCHER 1009, NCIC-BR-26. Results from another large-scale randomized trial (ARCHER 1050 comparing dacomitinib to gefitinib as first-line treatment of advanced treatment-naïve EGFR-mutant NSCLC patients will soon be available and will serve as the lynchpin trial for the potential approval of dacomitinib in NSCLC. Meanwhile, third-generation EGFR TKIs (eg, CO-1686 [rociletinib], AZ9291, HM61713, EGF816, and ASP8273 that preferentially

  3. Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

    Energy Technology Data Exchange (ETDEWEB)

    Venkitachalam, Srividya; Chueh, Fu-Yu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States); Yu, Chao-Lan, E-mail: chaolan.yu@rosalindfranklin.edu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Lmo2 expression is elevated in Lck-transformed cells. Black-Right-Pointing-Pointer Both endogenous and exogenous Lck localize in the nucleus. Black-Right-Pointing-Pointer Nuclear Lck is active in Lck-transformed cells. Black-Right-Pointing-Pointer Lck binds to the promoter region of Lmo2 gene in vivo. Black-Right-Pointing-Pointer In contrast to JAK2, Lck does not increase histone H3 phosphorylation on Tyr 41. -- Abstract: LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

  4. Prognostic value of protein tyrosine kinase 6 (PTK6) for long-term survival of breast cancer patients.

    Science.gov (United States)

    Aubele, M; Walch, A K; Ludyga, N; Braselmann, H; Atkinson, M J; Luber, B; Auer, G; Tapio, S; Cooke, T; Bartlett, J M S

    2008-10-01

    The cytoplasmic tyrosine kinase PTK6 (BRK) shows elevated expression in approximately two-thirds of primary breast tumours, and is implicated in EGF receptor-dependent signalling and epithelial tumorigenesis. Using immunohistochemistry, we performed a retrospective study on 426 archival breast cancer samples from patients with long-term follow-up and compared the protein expression levels of PTK6, the HER receptors, Sam68 (a substrate of PTK6), and signalling proteins including MAP kinase (MAPK), phosphorylated MAPK (P-MAPK), and PTEN. We show that PTK6 expression is of significant prognostic value in the outcome of breast carcinomas. In multivariate analysis, the disease-free survival of patients of >or=240 months was directly associated with the protein expression level of PTK6 (P

  5. S-Nitrosylation of proline-rich tyrosine kinase 2 involves its activation induced by oxygen-glucose deprivation.

    Science.gov (United States)

    Yan, Xian-Liang; Liu, Dong-Hai; Zhang, Gong-Liang; Hu, Shu-Qun; Chen, Yu-Guo; Xu, Tie

    2015-06-15

    Previous studies have demonstrated that activation of proline-rich tyrosine kinase 2 (PYK2) in cerebral ischemia is involved in the modulation of N-methyl-d-aspartate-type (NMDA) glutamate receptor activity and Ca(2+) dynamics, resulting in ischemic neuron death ultimately. A number of reports indicate that PYK2 is a redox sensitive kinase that must be activated by an estrogen-induced reactive oxygen species (ROS). However, the mechanism of PYK2 activation remains incompletely illustrated. Accumulating attention is focused on nitric oxide (NO, a free radical) which plays a critical role in cellular signal transduction through stimulus-coupled S-nitrosylation of cysteine residues. Here we reported that PYK2 over-expressed in human embryonic kidney (HEK293) cells was S-nitrosylated (forming SNO-PYK2) by reacting with GSNO, an exogenous NO donor, at one critical cysteine residue (Cys534) with a biotin switch assay. Moreover, our results showed that S-nitrosylation and phosphorylation of PYK2 over-expressed in SH-SY5Y cells was significantly increased after oxygen-glucose deprivation (OGD). We further investigated whether the activation (phosphorylation) of PYK2 was associated with S-nitrosylation following SH-SY5Y cells OGD. Our results showed that the cysteine534 residue (site of S-nitrosylation) mutant PYK2 over-expressed in SH-SY5Y cells diminished S-nitrosylation of PYK2 and inhibited its phosphorylation induced by OGD. In addition, overexpression of the mutant PYK2 protein could prevent nuclear accumulation and abrogate neuronal cell death compared to wild type PYK2 in SH-SY5Y cells induced by OGD. These data suggest that the activation of PYK2 following OGD may be modulated by S-nitrosylation, which provides a new avenue for stroke therapy by targeting the post-translational modification machinery.

  6. TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations

    Science.gov (United States)

    Castoldi, Raffaella; Schanzer, Jürgen; Panke, Christian; Jucknischke, Ute; Neubert, Natalie J.; Croasdale, Rebecca; Scheuer, Werner; Auer, Johannes; Klein, Christian; Niederfellner, Gerhard; Kobold, Sebastian; Sustmann, Claudio

    2016-01-01

    Monoclonal antibody-based targeted tumor therapy has greatly improved treatment options for patients. Antibodies against oncogenic receptor tyrosine kinases (RTKs), especially the ErbB receptor family, are prominent examples. However, long-term efficacy of such antibodies is limited by resistance mechanisms. Tumor evasion by a priori or acquired activation of other kinases is often causative for this phenomenon. These findings led to an increasing number of combination approaches either within a protein family, e.g. the ErbB family or by targeting RTKs of different phylogenetic origin like HER1 and cMet or HER1 and IGF1R. Progress in antibody engineering technology enabled generation of clinical grade bispecific antibodies (BsAbs) to design drugs inherently addressing such resistance mechanisms. Limited data are available on multi-specific antibodies targeting three or more RTKs. In the present study, we have evaluated the cloning, eukaryotic expression and purification of tetraspecific, tetravalent Fc-containing antibodies targeting HER3, cMet, HER1 and IGF1R. The antibodies are based on the combination of single-chain Fab and Fv fragments in an IgG1 antibody format enhanced by the knob-into-hole technology. They are non-agonistic and inhibit tumor cell growth comparable to the combination of four parental antibodies. Importantly, TetraMabs show improved apoptosis induction and tumor growth inhibition over individual monospecific or BsAbs in cellular assays. In addition, a mimicry assay to reflect heterogeneous expression of antigens in a tumor mass was established. With this novel in vitro assay, we can demonstrate the superiority of a tetraspecific antibody to bispecific tumor antigen-binding antibodies in early pre-clinical development. PMID:27578890

  7. Molecular dissection of egg fertilization signaling with the aid of tyrosine kinase-specific inhibitor and activator strategies.

    Science.gov (United States)

    Sato, Ken-ichi; Iwasaki, Tetsushi; Hirahara, Shino; Nishihira, Yusuke; Fukami, Yasuo

    2004-03-11

    Fertilization is triggered by sperm-egg interaction and fusion that initiate a transient rise(s) in the free intracellular calcium ([Ca(2+)](i)) that is responsible for a series of biochemical and cell biological events, so-called "egg activation". Calcium-dependent egg activation leads to the initiation of developmental program that culminates in the birth of individuals. A growing body of knowledge has uncovered the molecular mechanisms underlying sperm-induced transient [Ca(2+)](i) increase(s) to some extent; namely, in most animals so far studied, a second messenger inositol 1,4,5-trisphosphate (IP(3)) seems to play a pivotal role in inducing [Ca(2+)](i) transient(s) at fertilization. However, signaling mechanisms used by sperm to initiate IP(3)-[Ca(2+)](i) transient pathway have not been elucidated. To approach this problem, we have employed African clawed frog, Xenopus laevis, as a model animal and conducted experiments designed specifically to determine the role of the Src family protein-tyrosine kinases (SFKs or Src family PTKs) in the sperm-induced egg activation. This review compiles information about the use of PTK-specific inhibitors and activators for analyzing signal transduction events in egg fertilization. Specifically, we focus on molecular identification of Xenopus Src and the signaling mechanism of the Src-dependent egg activation that has been established recently. We also summarize recent advances in understanding the role of the Src family kinases in egg fertilization of other model organisms, and discuss future directions of the field.

  8. Association study of neurotrophic tyrosine kinase receptor type 2 (NTRK2) and childhood-onset mood disorders.

    Science.gov (United States)

    Adams, Jennifer H; Wigg, Karen G; King, Nicole; Burcescu, Irina; Vetró, Agnes; Kiss, Eniko; Baji, Ildikó; George, Charles J; Kennedy, James L; Kovacs, Maria; Barr, Cathy L

    2005-01-05

    Childhood-onset mood disorders (COMD) are often familial, and twin studies of COMD provide compelling evidence that genetic factors are involved. Deficits in neural plasticity have been suggested to underlie the development of depression. The receptor tropomyosin related kinase B (TrkB) and its ligand, brain derived neurotrophic factor (BDNF), play essential roles in neural plasticity, and mRNA expression of both of these genes has been shown to be influenced by stress and chronic antidepressant treatment. In addition, TrkB knock-out mice display inappropriate stress coping mechanisms. Having previously shown that BDNF is associated with COMD, in this study we investigated the gene encoding TrkB, neurotrophic tyrosine kinase, receptor, type 2 (NTRK2) as a susceptibility factor in COMD. We tested for association of NTRK2 with COMD in two independent samples: (a) a case-control sample matched on ethnicity and gender, consisting of 120 cases who met DSM III/IV criteria for major depressive or dysthymic disorder before age 14 or bipolar I/II before the age of 18, and controls, and (b) a family based control sample of 113 families collected in Hungary, identified by a proband between the age of 7 and 14 who met DSM IV criteria for major depressive disorder or bipolar I/II disorder. There was no evidence for an allelic or genotypic association of three polymorphisms of NTRK2 with COMD in the case-control sample. Also, in the family based sample, using the transmission disequilibrium test (TDT), we did not identify any evidence of allelic association for each marker individually or when haplotypes were analyzed. Based on these results, using these three polymorphisms, we do not find support for NTRK2 as a susceptibility gene for COMD.

  9. The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling.

    Science.gov (United States)

    Hikasa, Hiroki; Shibata, Mikihito; Hiratani, Ichiro; Taira, Masanori

    2002-11-01

    The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.

  10. Diagnosis of preeclampsia with soluble Fms-like tyrosine kinase 1/placental growth factor ratio

    DEFF Research Database (Denmark)

    Andersen, Louise Bjørkholt; Frederiksen-Møller, Britta; Work Havelund, Kathrine;

    2015-01-01

    The angiogenic factor ratio soluble Fms-kinase 1 (sFlt-1)/placental growth factor (PlGF) is a novel diagnostic tool for preeclampsia. We compared the efficacy of the KRYPTOR (BRAHMS) automated assays for sFlt-1 and PlGF with the Elecsys (Roche) assays in a routine clinical setting. Preeclamptic...... = .047). For early-onset preeclampsia, KRYPTOR area under the curve increased to 0.929 with a 100% specificity for preeclampsia at cut-off 85 and an 88.9% sensitivity for preeclampsia at cut-off 33. For women with preeclampsia and preterm delivery or Hemolysis, Elevated Liver enzymes, Low Platelet count...... (HELLP) syndrome, the KRYPTOR sFlt-1/PlGF ratio was manifold increased (P preeclampsia, preeclampsia with preterm delivery or HELLP, and among non-obese women....

  11. Effect of propofol on brain-derived neurotrophic factor and tyrosine kinase receptor B in the hippocampus of aged rats with chronic cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Gang Chen; Qiang Fu; Jiangbei Cao; Weidong Mi

    2012-01-01

    We intraperitoneally injected 10 and 50 mg/kg of propofol for 7 consecutive days to treat a rat model of chronic cerebral ischemia. A low-dose of propofol promoted the expression of brain-derived neurotrophic factor, tyrosine kinase receptor B, phosphorylated cAMP response element binding protein, and cAMP in the hippocampus of aged rats with chronic cerebral ischemia, but a high-dose of propofol inhibited their expression. Results indicated that the protective effect of propofol against cerebral ischemia in aged rats is related to changes in the expression of brain-derived neurotrophic factor and tyrosine kinase receptor B in the hippocampus, and that the cAMP-cAMP responsive element binding protein pathway is involved in the regulatory effect of propofol on brain-derived neurotrophic factor expression.

  12. A receptor tyrosine kinase, UFO/Axl, and other genes isolated by a modified differential display PCR are overexpressed in metastatic prostatic carcinoma cell line DU145.

    Science.gov (United States)

    Jacob, A N; Kalapurakal, J; Davidson, W R; Kandpal, G; Dunson, N; Prashar, Y; Kandpal, R P

    1999-01-01

    We have used a modified differential display PCR protocol for isolating 3' restriction fragments of cDNAs specifically expressed or overexpressed in metastatic prostate carcinoma cell line DU145. Several cDNA fragments were identified that matched to milk fat globule protein, UFO/Axl, a receptor tyrosine kinase, human homologue of a Xenopus maternal transcript, laminin and laminin receptor, human carcinoma-associated antigen, and some expressed sequence tags. The transcript for milk fat globule protein, a marker protein shown to be overexpressed in breast tumors, was elevated in DU145 cells. The expression of UFO/Axl, a receptor tyrosine kinase, was considerably higher in DU145 cells as compared to normal prostate cells and prostatic carcinoma cell line PC-3. The overexpression of UFO oncogene in DU145 cells is discussed in the context of prostate cancer metastasis.

  13. A method to quantify several tyrosine kinase inhibitors in plasma by micellar liquid chromatography and validation according to the European Medicines Agency guidelines.

    Science.gov (United States)

    Garrido-Cano, Iris; García-García, Aurelio; Peris-Vicente, Juan; Ochoa-Aranda, Enrique; Esteve-Romero, Josep

    2015-11-01

    A procedure based on micellar liquid chromatography has been developed to monitor five tyrosine kinase inhibitors in plasma, prescribed against several kinds of cancer: erlotinib, imatinib, sunitinib, sorafenib and lapatinib. The sample was diluted in a micellar solution and directly injected, thus clean-up steps were not required. The analytes were resolved without interferences in 0.990), limit of detection (15-35 ng/mL), carry-over effect, accuracy (-10.4 to +11.0%), precision (<9.2%), matrix effect, robustness (<8.4%) and stability. The procedure is rapid, easy-to-handle, uses a low amount of toxic chemical provide reliable results. Finally, the method was successfully used to analyze the studied tyrosine kinase inhibitors in plasma from cancer patients.

  14. ANKRD54 preferentially selects Bruton’s Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library

    Science.gov (United States)

    Mohammad, Dara K.; Ylösmäki, Erkko; Choi, Hyunseok; Shrestha, Subhash; Wang, Qing; Nore, Beston F.; Saksela, Kalle; Smith, C. I. Edvard

    2017-01-01

    Bruton’s Tyrosine Kinase (BTK) is a cytoplasmic protein tyrosine kinase with a fundamental role in B-lymphocyte development and activation. The nucleocytoplasmic shuttling of BTK is specifically modulated by the Ankyrin Repeat Domain 54 (ANKRD54) protein and the interaction is known to be exclusively SH3-dependent. To identify the spectrum of the ANKRD54 SH3-interactome, we applied phage-display screening of a library containing all the 296 human SH3 domains. The BTK-SH3 domain was the prime interactor. Quantitative western blotting analysis demonstrated the accuracy of the screening procedure. Revealing the spectrum and specificity of ANKRD54-interactome is a critical step toward functional analysis in cells and tissues. PMID:28369144

  15. Study of Mutation in Tyrosine Protein Kinase of Insulin Receptor Gene in Patients with Polycystic Ovarian Syndrome

    Institute of Scientific and Technical Information of China (English)

    Min LI; Hong-yu QIU; Yong-yu SUN; Hong-fa LI; Yong-li CHU

    2003-01-01

    Objective To explore the molecular mechanism of insulin resistance in the patients with polycystic ovarian syndrome (PCOS)Methods Polymerase chain reaction, silver staining-single strand conformation polymorphism(PCR-SSCP) and DNA direct sequencing were used to detect the mutation of insulin receptor(INSR) gene in exon 17~21 with the abdominal wall adipose tissue from 31 patients with PCOS (PCOS Group) and 30 patients with pure hysteromyoma in reproductive lift (Control Group).Results Twenty-two variant SSCP patterns in exon 17 of INSR gene were detected. Direct sequence analysis of exon 17 showed that homozygous nonsense mutation was two alleles single nucleotide polymorphism(SNP) at the codon 1058 (CAC→CAT). Exons 18~21 were not detected with any significantly mutation. The INSR gene His1058C→T substitution collecting rate and insulin resistance were significantly higher in the PCOS group than in the control group (P=0.0293, P<0.05, P<0.01).Conclusion It is suggested that the SNP in codon 1058 of the INSR gene might be related with the insulin resistance in PCOS patients, which has hereditary tendency. And the missense mutation,nonsense mutation and frameshift mutation at exons 18~21 in tyrosine protein kinase region of INSR gene for PCOS patients were not frequently observed.

  16. Two cases of gastrointestinal perforation after radiotherapy in patients receiving tyrosine kinase inhibitor for advanced renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Inoue Takaaki

    2012-08-01

    Full Text Available Abstract We report two cases of gastrointestinal perforation (GIP after radiotherapy in patients receiving tyrosine kinase inhibitor (TKI for advanced renal cell carcinoma (RCC. Case 1 was a 61-year-old woman with lung metastases after a radical nephrectomy for a right RCC (cT3aN0M0 treated with interferon-alpha (OIF, 5 MIU, three times per week. She developed lytic metastases of the left femur and the left acetabulum. She was treated with palliative radiotherapy to the metastatic portion (3 Gy × 10 fractions, and 400 mg sorafenib twice per day plus continuing interferon alpha. She experienced sudden left lower abdominal pain after four weeks of treatment, and was diagnosed with a perforation of the sigmoid colon with fecal peritonitis. Case 2 was a 48-year-old man with lung, lymph node, and bone metastases after a radical nephrectomy for a right RCC (cT2N0M0, and was treated with 400 mg sorafenib twice per day. He developed lytic bone metastases of the lumbar vertebrae, which was treated with palliative radiotherapy to L2-4 (3 Gy × 10 fractions. He experienced sudden abdominal pain after two months of radiation treatment, and was diagnosed with a perforation of the sigmoid colon with fecal peritonitis. These cases underwent radiotherapy, and therefore this may be related to the radiosensitivity of TKI.

  17. Antitumor and antiangiogenic effect of the dual EGFR and HER-2 tyrosine kinase inhibitor lapatinib in a lung cancer model

    Directory of Open Access Journals (Sweden)

    Collantes Maria

    2010-05-01

    Full Text Available Abstract Background There is strong evidence demonstrating that activation of epidermal growth factor receptors (EGFRs leads to tumor growth, progression, invasion and metastasis. Erlotinib and gefitinib, two EGFR-targeted agents, have been shown to be relevant drugs for lung cancer treatment. Recent studies demonstrate that lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER-2 receptors, is clinically effective against HER-2-overexpressing metastatic breast cancer. In this report, we investigated the activity of lapatinib against non-small cell lung cancer (NSCLC. Methods We selected the lung cancer cell line A549, which harbors genomic amplification of EGFR and HER-2. Proliferation, cell cycle analysis, clonogenic assays, and signaling cascade analyses (by western blot were performed in vitro. In vivo experiments with A549 cells xenotransplanted into nude mice treated with lapatinib (with or without radiotherapy were also carried out. Results Lapatinib dramatically reduced cell proliferation (P P P in vitro. Furthermore, lapatinib induced G1 cell cycle arrest (P P In vivo experiments revealed that A549 tumor-bearing mice treated with lapatinib had significantly less active tumors (as assessed by PET analysis (P P Conclusion Overall, these data suggest that lapatinib may be a clinically useful agent for the treatment of lung cancer.

  18. Characterization and response of newly developed high-grade glioma cultures to the tyrosine kinase inhibitors, erlotinib, gefitinib and imatinib.

    LENUS (Irish Health Repository)

    Kinsella, Paula

    2012-03-10

    High-grade gliomas (HGG), are the most common aggressive brain tumours in adults. Inhibitors targeting growth factor signalling pathways in glioma have shown a low clinical response rate. To accurately evaluate response to targeted therapies further in vitro studies are necessary. Growth factor pathway expression using epidermal growth factor receptor (EGFR), mutant EGFR (EGFRvIII), platelet derived growth factor receptor (PDGFR), C-Kit and C-Abl together with phosphatase and tensin homolog (PTEN) expression and downstream activation of AKT and phosphorylated ribosomal protein S6 (P70S6K) was analysed in 26 primary glioma cultures treated with the tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib and imatinib. Response to TKIs was assessed using 50% inhibitory concentrations (IC(50)). Response for each culture was compared with the EGFR\\/PDGFR immunocytochemical pathway profile using hierarchical cluster analysis (HCA) and principal component analysis (PCA). Erlotinib response was not strongly associated with high expression of the growth factor pathway components. PTEN expression did not correlate with response to any of the three TKIs. Increased EGFR expression was associated with gefitinib response; increased PDGFR-α expression was associated with imatinib response. The results of this in vitro study suggest gefitinib and imatinib may have therapeutic potential in HGG tumours with a corresponding growth factor receptor expression profile.

  19. An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment

    Directory of Open Access Journals (Sweden)

    Tiziana Grafone

    2012-04-01

    Full Text Available Hematopoiesis, the process by which the hematopoietic stem cells and progenitors differentiate into blood cells of various lineages, involves complex interactions of transcription factors that modulate the expression of downstream genes and mediate proliferation and differentiation signals. Despite the many controls that regulate hematopoiesis, mutations in the regulatory genes capable of promoting leukemogenesis may occur. The FLT3 gene encodes a tyrosine kinase receptor that plays a key role in controlling survival, proliferation and differentiation of hematopoietic cells. Mutations in this gene are critical in causing a deregulation of the delicate balance between cell proliferation and differentiation. In this review, we provide an update on the structure, synthesis and activation of the FLT3 receptor and the subsequent activation of multiple downstream signaling pathways. We also review activating FLT3 mutations that are frequently identified in acute myeloid leukemia, cause activation of more complex downstream signaling pathways and promote leukemogenesis. Finally, FLT3 has emerged as an important target for molecular therapy. We, therefore, report on some recent therapies directed against it.

  20. Consequences of two naturally occurring missense mutations in the structure and function of Bruton agammaglobulinemia tyrosine kinase.

    Science.gov (United States)

    Vargas-Hernández, Alexander; López-Herrera, Gabriela; Maravillas-Montero, José L; Vences-Catalán, Felipe; Mogica-Martínez, Dolores; Rojo-Domínguez, Arturo; Espinosa-Rosales, Francisco J; Santos-Argumedo, Leopoldo

    2012-04-01

    Bruton agammaglobulinemia tyrosine kinase (BTK) is a key protein in the B-cell receptor (BCR) signaling pathway and plays an essential role in the differentiation of B lymphocytes. X-linked agammaglobulinemia (XLA) is a primary humoral immunodeficiency caused by mutations in the gene encoding BTK. Previously, we identified two novel variations, L111P and E605G, in BTK; these are localized within the pleckstrin homology and Src homology 1 domains, respectively. In the present study, we evaluated the potential effects of these variations on the structural conformation and the function of BTK. Using in silico methods, we found that the L111P and E650G variations are not located directly in protein-protein interfaces but close to them. They distorted the native structural conformation of the BTK protein, affecting not only its geometry and stability but also its ability for protein recognition and in consequence its functionality. To confirm the results of the in silico assays, WT BTK, L111P, and E650G variants were expressed in the BTK-deficient DT40 cell line. The mutant proteins exhibited an absence of catalytic activity, aberrant redistribution after BCR-crosslinking, and deficient intracellular calcium mobilization. This work demonstrates that L111 and E605 residues are fundamental for the activation and function of BTK.

  1. Neutrophil development and function critically depend on Bruton tyrosine kinase in a mouse model of X-linked agammaglobulinemia.

    Science.gov (United States)

    Fiedler, Katja; Sindrilaru, Anca; Terszowski, Grzegorz; Kokai, Enikö; Feyerabend, Thorsten B; Bullinger, Lars; Rodewald, Hans-Reimer; Brunner, Cornelia

    2011-01-27

    Bruton tyrosine kinase (Btk) is essential for B cell development and function and also appears to be important for myeloid cells. The bone marrow of Btk-deficient mice shows enhanced granulopoiesis compared with that of wild-type mice. In purified granulocyte-monocyte-progenitors (GMP) from Btk-deficient mice, the development of granulocytes is favored at the expense of monocytes. However, Btk-deficient neutrophils are impaired in maturation and function. Using bone marrow chimeras, we show that this defect is cell-intrinsic to neutrophils. In GMP and neutrophils, Btk plays a role in GM-CSF- and Toll-like receptor-induced differentiation. Molecular analyses revealed that expression of the lineage-determining transcription factors C/EBPα, C/EBPβ, and PU.1, depends on Btk. In addition, expression of several granule proteins, including myeloperoxidase, neutrophilic granule protein, gelatinase and neutrophil elastase, is Btk-dependent. In the Arthus reaction, an acute inflammatory response, neutrophil migration into tissues, edema formation, and hemorrhage are significantly reduced in Btk-deficient animals. Together, our findings implicate Btk as an important regulator of neutrophilic granulocyte maturation and function in vivo.

  2. p130Cas Couples the tyrosine kinase Bmx/Etk with regulation of the actin cytoskeleton and cell migration.

    Science.gov (United States)

    Abassi, Yama A; Rehn, Marko; Ekman, Niklas; Alitalo, Kari; Vuori, Kristiina

    2003-09-12

    Bmx/Etk, a member of the Tec/Btk family of nonreceptor kinases, has recently been shown to mediate cell motility in signaling pathways that become activated upon integrin-mediated cell adhesion (Chen, R., Kim, O., Li, M., Xiong, X., Guan, J. L., Kung, H. J., Chen, H., Shimizu, Y., and Qiu, Y. (2001) Nat Cell Biol. 3, 439-444). The molecular mechanisms of Bmx-induced cell motility have so far remained unknown. Previous studies by us and others have demonstrated that a complex formation between the docking protein p130Cas (Cas) and the adapter protein Crk is instrumental in connecting several stimuli to the regulation of actin cytoskeleton and cell motility. We demonstrate here that expression of Bmx leads to an interaction between Bmx and Cas at membrane ruffles, which are sites of active actin remodeling in motile cells. Expression of Bmx also enhances tyrosine phosphorylation of Cas and Cas.Crk complex formation, and coexpression of Bmx with Cas results in an enhanced membrane ruffling and haptotactic cell migration. Importantly, a mutant form of Bmx that fails to interact with Cas also fails to induce cell migration. Furthermore, expression of a dominant-negative form of Cas that is incapable of interacting with Crk inhibits Bmx-induced membrane ruffling and cell migration. These studies suggest that Bmx-Cas interaction, phosphorylation of Cas by Bmx, and subsequent Cas.Crk complex formation functionally couple Bmx to the regulation of actin cytoskeleton and cell motility.

  3. Tyrosine kinase ETK/BMX is up-regulated in bladder cancer and predicts poor prognosis in patients with cystectomy.

    Science.gov (United States)

    Guo, Shengjie; Sun, Feng; Guo, Zhiyong; Li, Wei; Alfano, Alan; Chen, Hegang; Magyar, Clara E; Huang, Jiaoti; Chai, Toby C; Qiu, Shaopeng; Qiu, Yun

    2011-03-07

    Deregulation of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors. In this report, we demonstrated that ETK expression is progressively increased during bladder cancer progression. We found that down-regulation of ETK in bladder cancer cells attenuated STAT3 and AKT activity whereas exogenous overexpression of ETK had opposite effects, suggesting that deregulation of ETK may attribute to the elevated activity of STAT3 and AKT frequently detected in bladder cancer. The survival, migration and invasion of bladder cancer cells were significantly compromised when ETK expression was knocked down by a specific shRNA. In addition, we showed that ETK localizes to mitochondria in bladder cancer cells through interacting with Bcl-XL and regulating ROS production and drug sensitivity. Therefore, ETK may play an important role in regulating survival, migration and invasion by modulating multiple signaling pathways in bladder cancer cells. Immunohistochemistry analysis on tissue microarrays containing 619 human bladder tissue samples shows that ETK is significantly upregulated during bladder cancer development and progression and ETK expression level predicts the survival rate of patients with cystectomy. Taken together, our results suggest that ETK may potentially serve as a new drug target for bladder cancer treatment as well as a biomarker which could be used to identify patients with higher mortality risk, who may be benefited from therapeutics targeting ETK activity.

  4. Tyrosine kinase ETK/BMX is up-regulated in bladder cancer and predicts poor prognosis in patients with cystectomy.

    Directory of Open Access Journals (Sweden)

    Shengjie Guo

    Full Text Available Deregulation of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors. In this report, we demonstrated that ETK expression is progressively increased during bladder cancer progression. We found that down-regulation of ETK in bladder cancer cells attenuated STAT3 and AKT activity whereas exogenous overexpression of ETK had opposite effects, suggesting that deregulation of ETK may attribute to the elevated activity of STAT3 and AKT frequently detected in bladder cancer. The survival, migration and invasion of bladder cancer cells were significantly compromised when ETK expression was knocked down by a specific shRNA. In addition, we showed that ETK localizes to mitochondria in bladder cancer cells through interacting with Bcl-XL and regulating ROS production and drug sensitivity. Therefore, ETK may play an important role in regulating survival, migration and invasion by modulating multiple signaling pathways in bladder cancer cells. Immunohistochemistry analysis on tissue microarrays containing 619 human bladder tissue samples shows that ETK is significantly upregulated during bladder cancer development and progression and ETK expression level predicts the survival rate of patients with cystectomy. Taken together, our results suggest that ETK may potentially serve as a new drug target for bladder cancer treatment as well as a biomarker which could be used to identify patients with higher mortality risk, who may be benefited from therapeutics targeting ETK activity.

  5. Overcoming the drug resistance problem with second-generation tyrosine kinase inhibitors: from enzymology to structural models.

    Science.gov (United States)

    Crespan, E; Zucca, E; Maga, G

    2011-01-01

    Protein phosphorylation is one of the major pathways used by eukaryotic cells to propagate signals to the final effectors, regulating multiple aspects of the living cell, such as metabolism, growth, differentiation, adhesion, motility, genome stability and death. In this context, tyrosine kinases (TKs) play a central role in signal transduction and their overexpression or disregulated activity has been implicated in tumor onset and malignancy progression. To date, eight TKs inhibitors have been approved by FDA for the treatment of specific tumors. In spite of their efficacy, insurgence of resistance is a common feature after prolonged administration. The selective pressure by these drugs, in fact, induces clonal expansion of subsets of cancer cells harboring TKs mutations, leading to decreased inhibition potency. Alternatively, resistance to TK inhibitors can be acquired through the activation of others, often unrelated, TKs. For this reason, while stringent target selectivity of TKs inhibitors has been always considered a desirable feature in order to limit toxicity, molecules targeting different TKs have been recently shown to be promising anti-cancer agents as well. Understanding the molecular mechanisms that confer resistance to TK inhibitors, through a combination of enzymatic, structural and cellular studies, is essential in the development of second generation inhibitors active also towards drug resistant tumors.

  6. Tyrosine kinase inhibitor BIBF1120 ameliorates inflammation, angiogenesis and fibrosis in CCl4-induced liver fibrogenesis mouse model

    Science.gov (United States)

    Öztürk Akcora, Büsra; Storm, Gert; Prakash, Jai; Bansal, Ruchi

    2017-01-01

    Hepatic fibrosis, a progressive chronic disease mainly caused by hepatitis viral infections, alcohol abuse or metabolic syndrome leading to liver dysfunction and is the growing cause of mortality worldwide. Tyrosine kinase inhibitor BIBF1120 (Nintedanib) has been evaluated in clinical trials for idiopathic pulmonary fibrosis and advanced Hepatocellular carcinoma, but has not been explored for liver fibrosis yet. In this study, we aimed to investigate the therapeutic effects and mechanism of BIBF1120 in liver fibrogenesis. The effects of BIBF1120 were evaluated in TGFβ-activated mouse 3T3 fibroblasts, LX2 cells, primary human hepatic stellate cells (HSCs) and CCl4-induced liver fibrogenesis mouse model. Fibroblasts-conditioned medium studies were performed to assess the paracrine effects on macrophages and endothelial cells. In-vitro in TGFβ-activated fibroblasts, BIBF1120 significantly inhibited expression of major fibrotic parameters, wound-healing and contractility. In vivo in CCl4-induced acute liver injury model, post-disease BIBF1120 administration significantly attenuated collagen accumulation and HSC activation. Interestingly, BIBF1120 drastically inhibited intrahepatic inflammation and angiogenesis. To further elucidate the mechanism of action, 3T3-conditioned medium studies demonstrated increased 3T3-mediated macrophage chemotaxis and endothelial cells tube formation and activation, which was significantly decreased by BIBF1120. These results suggests that BIBF1120 can be a potential therapeutic approach for the treatment of liver fibrosis. PMID:28291245

  7. LC-MS/MS assay for the quantitation of the tyrosine kinase inhibitor neratinib in human plasma.

    Science.gov (United States)

    Kiesel, Brian F; Parise, Robert A; Wong, Alvin; Keyvanjah, Kiana; Jacobs, Samuel; Beumer, Jan H

    2017-02-05

    Neratinib is an orally available tyrosine kinase inhibitor targeting HER2 (ERBB2) and EGFR (ERBB). It is being clinically evaluated for the treatment of breast and other solid tumors types as a single agent or in combination with other chemotherapies. In support of several phase I/II clinical trials investigating neratinib combinations, we developed and validated a novel LC-MS/MS assay for the quantification of neratinib in 100μL of human plasma with a stable isotopic internal standard. Analytes were extracted from plasma using protein precipitation and evaporation of the resulting supernatant followed by resuspension. Chromatographic separation was achieved using an Acquity UPLC BEH Shield RP18 column and a gradient methanol-water mobile phase containing 10% ammonium acetate. An ABI 4000 mass spectrometer and electrospray positive mode ionization were used for detection. The assay was linear from 2 to 1,000ng/mL and proved to be accurate (98.9-106.5%) and precise (<6.2%CV), and met the FDA guidance for bioanalytical method validation. This LC-MS/MS assay will be an essential tool to further define the pharmacokinetics of neratinib.

  8. Met receptor tyrosine kinase signaling induces secretion of the angiogenic chemokine interleukin-8/CXCL8 in pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Kristen S Hill

    Full Text Available At diagnosis, the majority of pancreatic cancer patients present with advanced disease when curative resection is no longer feasible and current therapeutic treatments are largely ineffective. An improved understanding of molecular targets for effective intervention of pancreatic cancer is thus urgent. The Met receptor tyrosine kinase is one candidate implicated in pancreatic cancer. Notably, Met is over expressed in up to 80% of invasive pancreatic cancers but not in normal ductal cells correlating with poor overall patient survival and increased recurrence rates following surgical resection. However the functional role of Met signaling in pancreatic cancer remains poorly understood. Here we used RNA interference to directly examine the pathobiological importance of increased Met signaling for pancreatic cancer. We show that Met knockdown in pancreatic tumor cells results in decreased cell survival, cell invasion, and migration on collagen I in vitro. Using an orthotopic model for pancreatic cancer, we provide in vivo evidence that Met knockdown reduced tumor burden correlating with decreased cell survival and tumor angiogenesis, with minimal effect on cell growth. Notably, we report that Met signaling regulates the secretion of the pro-angiogenic chemokine interleukin-8/CXCL8. Our data showing that the interleukin-8 receptors CXCR1 and CXCR2 are not expressed on pancreatic tumor cells, suggests a paracrine mechanism by which Met signaling regulates interleukin-8 secretion to remodel the tumor microenvironment, a novel finding that could have important clinical implications for improving the effectiveness of treatments for pancreatic cancer.

  9. PAH- and PCB-induced Alterations of Protein Tyrosine Kinase and Cytokine Gene Transcription in Harbor Seal (Phoca Vitulina PBMC

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2005-01-01

    Full Text Available Mechanisms underlying in vitro immunomodulatory effects of polycyclic aromatic hydrocarbons (PAHs and polychlorinated biphenyls (PCBs were investigated in harbor seal peripheral leukocytes, via real-time PCR. We examined the relative genetic expression of the protein tyrosine kinases (PTKs Fyn and Itk, which play a critical role in T cell activation, and IL-2, a cytokine of central importance in initiating adaptive immune responses. IL-1, the macrophage-derived pro-inflammatory cytokine of innate immunity, was also included as a measure of macrophage function. Harbor seal PBMC were exposed to the prototypic immunotoxic PAH benzo[a]pyrene (BaP, 3,3',4,4',5,5'-hexachlorobiphenyl (CB-169, a model immunotoxic PCB, or DMSO (vehicle control. Exposure of Con A-stimulated harbor seal PBMC to both BaP and CB-169 produced significantly altered expression in all four targets relative to vehicle controls. The PTKs Fyn and Itk were both up-regulated following exposure to BaP and CB-169. In contrast, transcripts for IL-2 and IL-1 were decreased relative to controls by both treatments. Our findings are consistent with those of previous researchers working with human and rodent systems and support a hypothesis of contaminant-altered lymphocyte function mediated (at least in part by disruption of T cell receptor (TCR signaling and cytokine production.

  10. Bruton's Tyrosine Kinase (BTK and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages.

    Directory of Open Access Journals (Sweden)

    Karin Strijbis

    Full Text Available Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.

  11. Serum Soluble Fms-Like Tyrosine Kinase 1 (sFlt-1) Predicts the Severity of Acute Pancreatitis

    Science.gov (United States)

    Dumnicka, Paulina; Sporek, Mateusz; Mazur-Laskowska, Małgorzata; Ceranowicz, Piotr; Kuźniewski, Marek; Drożdż, Ryszard; Ambroży, Tadeusz; Olszanecki, Rafał; Kuśnierz-Cabala, Beata

    2016-01-01

    Organ failure is the most important determinant of the severity of acute pancreatitis (AP). Soluble fms-like tyrosine kinase 1 (sFlt-1) is positively associated with organ failure in sepsis. Our aim was to evaluate the diagnostic utility of automated sFlt-1 measurements for early prediction of AP severity. Adult patients (66) with AP were recruited, including 46 with mild (MAP), 15 with moderately-severe (MSAP) and 5 with severe AP (SAP). Serum and urine samples were collected twice. Serum sFlt-1 was measured with automated electrochemiluminescence immunoassay. Serum concentrations of sFlt-1 were significantly higher in patients with MSAP and SAP as compared to MAP. SAP patients had the highest concentrations. At 24 and 48 h, sFlt-1 positively correlated with inflammatory markers (leukocyte count, C-reactive protein), kidney function (creatinine, urea, cystatin C, serum and urine neutrophil gelatinase-associated lipocalin, urine albumin/creatinine ratio), D-dimer and angiopoietin-2. sFlt-1 positively correlated with the bedside index of severity in AP (BISAP) score and the duration of hospital stay. Serum sFlt-1 above 139 pg/mL predicted more severe AP (MSAP + SAP). In the early phase of AP, sFlt-1 is positively associated with the severity of AP and predicts organ failure, in particular kidney failure. Serum sFlt-1 may be a practical way to improve early assessment of AP severity. PMID:27929426

  12. Ganoderma lucidum Combined with the EGFR Tyrosine Kinase Inhibitor, Erlotinib Synergize to Reduce Inflammatory Breast Cancer Progression.

    Science.gov (United States)

    Suárez-Arroyo, Ivette J; Rios-Fuller, Tiffany J; Feliz-Mosquea, Yismeilin R; Lacourt-Ventura, Mercedes; Leal-Alviarez, Daniel J; Maldonado-Martinez, Gerónimo; Cubano, Luis A; Martínez-Montemayor, Michelle M

    2016-01-01

    The high incidence of resistance to Tyrosine Kinase Inhibitors (TKIs) targeted against EGFR and downstream pathways has increased the necessity to identify agents that may be combined with these therapies to provide a sustained response for breast cancer patients. Here, we investigate the therapeutic potential of Ganoderma lucidum extract (GLE) in breast cancer, focusing on the regulation of the EGFR signaling cascade when treated with the EGFR TKI, Erlotinib. SUM-149, or intrinsic Erlotinib resistant MDA-MB-231 cells, and a successfully developed Erlotinib resistant cell line, rSUM-149 were treated with increasing concentrations of Erlotinib, GLE, or their combination (Erlotinib/GLE) for 72h. Treatment effects were tested on cell viability, cell proliferation, cell migration and invasion. To determine tumor progression, severe combined immunodeficient mice were injected with SUM-149 cells and then treated with Erlotinib/GLE or Erlotinib for 13 weeks. We assessed the protein expression of ERK1/2 and AKT in in vitro and in vivo models. Our results show that GLE synergizes with Erlotinib to sensitize SUM-149 cells to drug treatment, and overcomes intrinsic and developed Erlotinib resistance. Also, Erlotinib/GLE decreases SUM-149 cell viability, proliferation, migration and invasion. GLE increases Erlotinib sensitivity by inactivating AKT and ERK signaling pathways in our models. We conclude that a combinatorial therapeutic approach may be the best way to increase prognosis in breast cancer patients with EGFR overexpressing tumors.

  13. Prognostic role of lemur tyrosine kinase-3 germline polymorphisms in adjuvant gastric cancer in Japan and the United States.

    Science.gov (United States)

    Wakatsuki, Takeru; LaBonte, Melissa J; Bohanes, Pierre O; Zhang, Wu; Yang, Dongyun; Azuma, Mizutomo; Barzi, Afsaneh; Ning, Yan; Loupakis, Fotios; Saadat, Siamak; Volz, Nico; Stintzing, Sebastian; El-Khoueiry, Rita; Koizumi, Wasaburo; Watanabe, Masahiko; Shah, Manish; Stebbing, Justin; Giamas, Georgios; Lenz, Heinz-Josef

    2013-10-01

    Lemur tyrosine kinase-3 (LMTK3) was recently identified as an estrogen receptor (ER)-α modulator related to endocrine therapy resistance, and its polymorphisms rs9989661 (T>C) T/T genotype and rs8108419 (G>A) G/G or A/G genotype predicted improved outcomes in breast cancer. Because different predominant ER distributions link to breast and gastric cancer and little is known of the prognostic role of LMTK3 in gastric cancer, this study was carried out to clarify the prognostic role of these polymorphisms in gastric cancer. One-hundred and sixty-nine Japanese and 137 U.S. patients with localized gastric adenocarcinoma were enrolled. Genomic DNA was extracted from blood or tissue, and all samples were analyzed by PCR-based direct DNA sequencing. Overall, these polymorphisms were not associated with survival in both cohorts. When gender was considered, in multivariate analysis, harboring rs9989661 T/T genotype was associated with disease-free survival [HR, 4.37; 95% confidence interval (CI), 2.08-9.18; P role of these polymorphisms may be negative in gastric cancer. These findings suggest that the estrogen pathway may play a prognostic role in patients with gastric cancer but this may be dependent on the regional differences both in physiology and genetic alterations of gastric cancer.

  14. Inhibition of corneal neovascularization with new Tyrosine Kinase Inhibitors targeting vascular endothelial growth factor receptors: Sunitinib malate and Sorafenib

    Directory of Open Access Journals (Sweden)

    Delnia Arshadi

    2007-06-01

    Full Text Available Corneal neovascularization (NV is a significant, sight-threatening, complication of many ocular surface disorders. Presence of new vessels in cornea can compromise clarity and thus vision. The data supporting a causal role for vascular endothelial growth factor (VEGF in corneal NV are extensive. Inhibition of VEGF remains as a main strategy for treating corneal NV. There is a growing body of evidence that corneal NV can be reduced by using anti-VEGF agents. Sunitinib malate and Sorafenib are new orally bio-available anti-angiogenic agents undergoing tests of efficacy in the treatment of various types of cancers. The main mechanism of these drugs is inhibiting angiogenesis by diminishing signaling through VEGF receptor1 (VEGFR1, VEGFR2, and platelet-derived growth factor receptors. Since VEGF exerts its angiogenic effects through tyrosine kinase receptors in cornea, any mechanisms which reduce VEGF signaling may inhibit corneal NV or at least attenuate it. Based on this fact we herein hypothesize that Sunitinib malate and Sorafenib can be prepared in topical form and be used in corneal neovascularization states. These approaches offer new hope for the successful treatment of corneal NV. Further investigations in animal models are needed to place these two drugs alongside corneal NV therapeutics.

  15. Oncogenic activation of the Met receptor tyrosine kinase fusion protein, Tpr-Met, involves exclusion from the endocytic degradative pathway.

    Science.gov (United States)

    Mak, H H L; Peschard, P; Lin, T; Naujokas, M A; Zuo, D; Park, M

    2007-11-01

    Multiple mechanisms of dysregulation of receptor tyrosine kinases (RTKs) are observed in human cancers. In addition to gain-of-function, loss of negative regulation also contributes to oncogenic activation of RTKs. Negative regulation of many RTKs involves their internalization and degradation in the lysosome, a process regulated through ubiquitination. RTK oncoproteins activated following chromosomal translocation, are no longer transmembrane proteins, and are predicted to escape lysosomal degradation. To test this, we used the Tpr-Met oncogene, generated following chromosomal translocation of the hepatocyte growth factor receptor (Met). Unlike Met, Tpr-Met is localized in the cytoplasm and also lacks the binding site for Cbl ubiquitin ligases. We determined whether subcellular localization of Tpr-Met, and/or loss of its Cbl-binding site, is important for oncogenic activity. Presence of a Cbl-binding site and ubiquitination of cytosolic Tpr-Met oncoproteins does not alter their transforming activity. In contrast, plasma membrane targeting allows Tpr-Met to enter the endocytic pathway, and Tpr-Met transforming activity as well as protein stability are decreased in a Cbl-dependent manner. We show that transformation by Tpr-Met is in part dependent on its ability to escape normal downregulatory mechanisms. This provides a paradigm for many RTK oncoproteins activated following chromosomal translocation.

  16. Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation.

    Science.gov (United States)

    Rochman, M; Kartashov, A V; Caldwell, J M; Collins, M H; Stucke, E M; Kc, K; Sherrill, J D; Herren, J; Barski, A; Rothenberg, M E

    2015-07-01

    Although interleukin (IL)-13 and neurotrophins are functionally important for the pathogenesis of immune responses, the interaction of these pathways has not been explored. Herein, by interrogating IL-13-induced responses in human epithelial cells we show that neurotrophic tyrosine kinase receptor, type 1 (NTRK1), a cognate, high-affinity receptor for nerve growth factor (NGF), is an early transcriptional IL-13 target. Induction of NTRK1 was accompanied by accumulation of activating epigenetic marks in the promoter; transcriptional and epigenetic changes were signal transducer and activator of transcription 6 dependent. Using eosinophilic esophagitis as a model for human allergic inflammation, we found that NTRK1 was increased in inflamed tissue and dynamically expressed as a function of disease activity and that the downstream mediator of NTRK1 signaling early growth response 1 protein was elevated in allergic inflammatory tissue compared with control tissue. Unlike NTRK1, its ligand NGF was constitutively expressed in control and disease states, indicating that IL-13-stimulated NTRK1 induction is a limiting factor in pathway activation. In epithelial cells, NGF and IL-13 synergistically induced several target genes, including chemokine (C-C motif) ligand 26 (eotaxin-3). In summary, we have demonstrated that IL-13 confers epithelial cell responsiveness to NGF by regulating NTRK1 levels by a transcriptional and epigenetic mechanism and that this process likely contributes to allergic inflammation.

  17. Indirect comparisons of second-generation tyrosine kinase inhibitors in CML: case study using baseline population characteristics

    Directory of Open Access Journals (Sweden)

    Kimbach Tran Carpiuc

    2010-10-01

    Full Text Available Kimbach Tran Carpiuc1, Gianantonio Rosti2, Fausto Castagnetti2, Maarten Treur3, Jennifer Stephens11Pharmerit North America LLC, Bethesda, MD, USA; 2Department of Hematology and Oncology, S Orsola-Malpighi University Hospital, Bologna, Italy; 3Pharmerit Europe, Rotterdam, The NetherlandsAbstract: The use of indirect comparisons to evaluate the relative effectiveness between two or more treatments is widespread in the literature and continues to grow each year. Appropriate methodologies will be essential for integrating data from various published clinical trials into a systematic framework as part of the increasing emphasis on comparative effectiveness research. This article provides a case study example for clinicians using the baseline study population characteristics and response rates of the tyrosine kinase inhibitors in imatinib-resistant or imatinib-intolerant chronic myelogenous leukemia followed by a discussion of indirect comparison methods that are being increasingly implemented to address challenges with these types of comparisons.Keywords: comparative effectiveness research, meta-analysis, BCR–ABL-positive chronic myelogenous leukemia, imatinib mesylate, nilotinib, dasatinib 

  18. Involvement of receptor tyrosine kinase Tyro3 in amyloidogenic APP processing and β-amyloid deposition in Alzheimer's disease models.

    Directory of Open Access Journals (Sweden)

    Yan Zheng

    Full Text Available Alzheimer's disease (AD is the most common progressive neurodegenerative disease known to humankind. It is characterized by brain atrophy, extracellular amyloid plaques, and intracellular neurofibril tangles. β-Amyloid cascade is considered the major causative player in AD. Up until now, the mechanisms underlying the process of Aβ generation and accumulation in the brain have not been well understood. Tyro3 receptor belongs to the TAM receptor subfamily of receptor protein tyrosine kinases (RPTKs. It is specifically expressed in the neurons of the neocortex and hippocampus. In this study, we established a cell model stably expressing APPswe mutants and producing Aβ. We found that overexpression of Tyro3 receptor in the cell model significantly decreased Aβ generation and also down-regulated the expression of β-site amyloid precursor protein cleaving enzyme (BACE1. However, the effects of Tyro3 were inhibited by its natural ligand, Gas6, in a concentration-dependent manner. In order to confirm the role of Tyro3 in the progression of AD development, we generated an AD transgenic mouse model accompanied by Tyro3 knockdown. We observed a significant increase in the number of amyloid plaques in the hippocampus in the mouse model. More plaque-associated clusters of astroglia were also detected. The present study may help researchers determine the role of Tyro3 receptor in the neuropathology of AD.

  19. Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells.

    OpenAIRE

    1996-01-01

    mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutati...

  20. Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

    Science.gov (United States)

    Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

    2012-10-02

    Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

  1. Practical recommendation for rash and diarrhea management in Indian patients treated with tyrosine kinase inhibitors for the treatment of non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    P Parikh

    2016-01-01

    Full Text Available Tyrosine kinase inhibitors (TKIs are a pharmaceutical class of small molecules, orally available with manageable safety profile, approved worldwide for the treatment of several neoplasms, including lung, breast, kidney and pancreatic cancer as well as gastro-intestinal stromal tumours and chronic myeloid leukaemia. In recent years, management of lung cancer has been moving towards molecular-guided treatment, and the best example of this new approach is the use of the tyrosine kinase inhibitors (TKIs in patients with mutations in the epidermal growth factor receptor (EGFR. The identification of molecular predictors of response can allow the selection of patients who will be the most likely to respond to these tyrosine kinase inhibitors (TKIs. Gastrointestinal (GI adverse events (AEs are frequently observed in patients receiving EGFR tyrosine kinase inhibitor therapy and are most impactful on the patient's quality of life. Dermatologic side effects are also relatively common among patients treated with EGFR inhibitors. Evidence has emerged in recent years to suggest that the incidence and severity of rash, positively correlated with response to treatment.These skin disorders are generally mild or moderate in severity and can be managed by appropriate interventions or by reducing or interrupting the dose. Appropriate and timely management make it possible to continue a patient's quality of life and maintain compliance; however if these adverse events (AEs are not managed appropriately, and become more severe, treatment cessation may be warranted compromising clinical outcome. Strategies to improve the assessment and management of TKI related skin AEs are therefore essential to ensure compliance with TKI therapy, thereby enabling patients to achieve optimal benefits. This article provides a consensus on practical recommendation for the prevention and management of diarrhoea and rash in Non-Small Cell Lung Cancer (NSCLC patients receiving TKIs.

  2. The pan-HER family tyrosine kinase inhibitor afatinib overcomes HER3 ligand heregulin-mediated resistance to EGFR inhibitors in non-small cell lung cancer

    OpenAIRE

    Yonesaka, Kimio; KUDO, KEITA; Nishida, Satomi; Takahama, Takayuki; Iwasa, Tsutomu; Yoshida, Takeshi; Tanaka, Kaoru; Takeda, Masayuki; Kaneda, Hiroyasu; Okamoto, Isamu; Nishio, Kazuto; Nakagawa, Kazuhiko

    2015-01-01

    Afatinib is a second generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) characterized as an irreversible pan-human EGFR (HER) family inhibitor. Afatinib remains effective for a subpopulation of patients with non-small cell lung cancer (NSCLC) with acquired resistance to first generation EGFF-TKIs such as erlotinib. Heregulin activates HER3 in an autocrine fashion and causes erlotinib resistance in NSCLC. Here we examine whether afatinib is effective against hereg...

  3. The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel.

    OpenAIRE

    Fischer, H.; Machen, T E

    1996-01-01

    The role of the tyrosine kinase p60c-src on the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel was investigated with the cell-attached and excised patch clamp technique in conjunction with current noise analysis of recordings containing multiple channels per patch. Spectra of CFTR-generated current noise contained a low-frequency and a high-frequency Lorentzian noise component. In the cell-attached mode, the high-frequency Lorentzian was significantl...

  4. Multi-tyrosine kinase inhibitors in preclinical studies for pediatric CNS AT/RT: Evidence for synergy with Topoisomerase-I inhibition

    Directory of Open Access Journals (Sweden)

    Jayanthan Aarthi

    2011-12-01

    Full Text Available Abstract Background Currently, Atypical Teratoid Rhabdoid Tumor (AT/RT constitutes one of the most difficult to treat malignancies in pediatrics. Hence, new knowledge of potential targets for therapeutics and the development of novel treatment approaches are urgently needed. We have evaluated the presence of cytokine pathways and the effects of two clinically available multi-tyrosine kinase inhibitors for cytotoxicity, target modulation and drug combinability against AT/RT cell lines. Results AT/RT cell lines expressed measurable quantities of VEGF, FGF, PDGF and SDF-1, although the absolute amounts varied between the cell lines. The targeted receptor tyrosine kinase inhibitor sorafenib inhibited the key signaling molecule Erk, which was activated following the addition of own conditioned media, suggesting the existence of autocrine/paracrine growth stimulatory pathways. The multi-tyrosine kinase inhibitors sorafenib and sunitinib also showed significant growth inhibition of AT/RT cells and their activity was enhanced by combination with the topoisomerase inhibitor, irinotecan. The loss of cytoplasmic NF-kappa-B in response to irinotecan was diminished by sorafenib, providing evidence for a possible benefit for this drug combination. Conclusions In addition to previously described involvement of insulin like growth factor (IGF family of cytokines, a multitude of other growth factors may contribute to the growth and survival of AT/RT cells. However, consistent with the heterogeneous nature of this tumor, quantitative and qualitative differences may exist among different tumor samples. Multi-tyrosine kinase inhibitors appear to have effective antitumor activity against all cell lines studied. In addition, the target modulation studies and drug combinability data provide the groundwork for additional studies and support the evaluation of these agents in future treatment protocols.

  5. Autophosphorylation of the Bacterial Tyrosine-Kinase CpsD Connects Capsule Synthesis with the Cell Cycle in Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Julien Nourikyan

    2015-09-01

    Full Text Available Bacterial capsular polysaccharides (CPS are produced by a multi-protein membrane complex, in which a particular type of tyrosine-autokinases named BY-kinases, regulate their polymerization and export. However, our understanding of the role of BY-kinases in these processes remains incomplete. In the human pathogen Streptococcus pneumoniae, the BY-kinase CpsD localizes at the division site and participates in the proper assembly of the capsule. In this study, we show that the cytoplasmic C-terminal end of the transmembrane protein CpsC is required for CpsD autophosphorylation and localization at mid-cell. Importantly, we demonstrate that the CpsC/CpsD complex captures the polysaccharide polymerase CpsH at the division site. Together with the finding that capsule is not produced at the division site in cpsD and cpsC mutants, these data show that CPS production occurs exclusively at mid-cell and is tightly dependent on CpsD interaction with CpsC. Next, we have analyzed the impact of CpsD phosphorylation on CPS production. We show that dephosphorylation of CpsD induces defective capsule production at the septum together with aberrant cell elongation and nucleoid defects. We observe that the cell division protein FtsZ assembles and localizes properly although cell constriction is impaired. DAPI staining together with localization of the histone-like protein HlpA further show that chromosome replication and/or segregation is defective suggesting that CpsD autophosphorylation interferes with these processes thus resulting in cell constriction defects and cell elongation. We show that CpsD shares structural homology with ParA-like ATPases and that it interacts with the chromosome partitioning protein ParB. Total internal reflection fluorescence microscopy imaging demonstrates that CpsD phosphorylation modulates the mobility of ParB. These data support a model in which phosphorylation of CpsD acts as a signaling system coordinating CPS synthesis with

  6. Fully automated synthesis of (phosphopeptide arrays in microtiter plate wells provides efficient access to protein tyrosine kinase characterization

    Directory of Open Access Journals (Sweden)

    Goldstein David J

    2005-01-01

    Full Text Available Abstract Background Synthetic peptides have played a useful role in studies of protein kinase substrates and interaction domains. Synthetic peptide arrays and libraries, in particular, have accelerated the process. Several factors have hindered or limited the applicability of various techniques, such as the need for deconvolution of combinatorial libraries, the inability or impracticality of achieving full automation using two-dimensional or pin solid phases, the lack of convenient interfacing with standard analytical platforms, or the difficulty of compartmentalization of a planar surface when contact between assay components needs to be avoided. This paper describes a process for synthesis of peptides and phosphopeptides on microtiter plate wells that overcomes previous limitations and demonstrates utility in determination of the epitope of an autophosphorylation site phospho-motif antibody and utility in substrate utilization assays of the protein tyrosine kinase, p60c-src. Results The overall reproducibility of phospho-peptide synthesis and multiplexed EGF receptor (EGFR autophosphorylation site (pY1173 antibody ELISA (9H2 was within 5.5 to 8.0%. Mass spectrometric analyses of the released (phosphopeptides showed homogeneous peaks of the expected molecular weights. An overlapping peptide array of the complete EGFR cytoplasmic sequence revealed a high redundancy of 9H2 reactive sites. The eight reactive phospopeptides were structurally related and interestingly, the most conserved antibody reactive peptide motif coincided with a subset of other known EGFR autophosphorylation and SH2 binding motifs and an EGFR optimal substrate motif. Finally, peptides based on known substrate specificities of c-src and related enzymes were synthesized in microtiter plate array format and were phosphorylated by c-Src with the predicted specificities. The level of phosphorylation was proportional to c-Src concentration with sensitivities below 0.1 Units of

  7. Interleukins 2, 4, 7, and 15 stimulate tyrosine phosphorylation of insulin receptor substrates 1 and 2 in T cells. Potential role of JAK kinases.

    Science.gov (United States)

    Johnston, J A; Wang, L M; Hanson, E P; Sun, X J; White, M F; Oakes, S A; Pierce, J H; O'Shea, J J

    1995-12-01

    The signaling molecules insulin receptor substrate (IRS)-1 and the newly described IRS-2 (4PS) molecule are major insulin and interleukin 4 (IL-4)-dependent phosphoproteins. We report here that IL-2, IL-7, and IL-15, as well as IL-4, rapidly stimulate the tyrosine phosphorylation of IRS-1 and IRS-2 in human peripheral blood T cells, NK cells, and in lymphoid cell lines. In addition, we show that the Janus kinases, JAK1 and JAK3, associate with IRS-1 and IRS-2 in T cells. Coexpression studies demonstrate that these kinases can tyrosine-phosphorylate IRS-2, suggesting a possible mechanism by which cytokine receptors may induce the tyrosine phosphorylation of IRS-1 and IRS-2. We further demonstrate that the p85 subunit of phosphoinositol 3-kinase associates with IRS-1 in response to IL-2 and IL-4 in T cells. Therefore, these data indicate that IRS-1 and IRS-2 may have important roles in T lymphocyte activation not only in response to IL-4, but also in response to IL-2, IL-7, and IL-15.

  8. Motor activity affects adult skeletal muscle re-innervation acting via tyrosine kinase receptors.

    Science.gov (United States)

    Sartini, Stefano; Bartolini, Fanny; Ambrogini, Patrizia; Betti, Michele; Ciuffoli, Stefano; Lattanzi, Davide; Di Palma, Michael; Cuppini, Riccardo

    2013-05-01

    Recently, muscle expression of brain-derived neurotrophic factor (BDNF) mRNA and protein under activity control has been reported. BDNF is a neurotrophin known to be involved in axon sprouting in the CNS. Hence, we set out to study the effect of chronic treadmill mid-intensity running on adult rat muscle re-innervation, and to explore the involvement of BDNF and tropomyosin-related kinase (Trk) receptors. After nerve crush, muscle re-innervation was evaluated using intracellular recordings, tension recordings, immunostaining and Western blot analyses. An enhanced muscle multiple innervation was found in running rats that was fully reversed to control values blocking Trk receptors or interrupting the running activity. An increase in muscle multiple innervation was also found in sedentary rats treated with a selective TrkB receptor agonist. The expression of TrkB receptors by intramuscular axons was demonstrated, and increased muscle expression of BDNF was found in running animals. The increase in muscle multiple innervation was consistent with the faster muscle re-innervation that we found in running animals. We conclude that, when regenerating axons contact muscle cells, muscle activity progressively increases modulating BDNF and possibly other growth factors, which in turn, acting via Trk receptors, induce axon sprouting to re-innervate skeletal muscle.

  9. Evaluation of placental growth factor and soluble Fms-like tyrosine kinase 1 as predictors of all-cause and cardiovascular mortality in patients with Type 1 diabetes with and without diabetic nephropathy

    DEFF Research Database (Denmark)

    Theilade, S; Lajer, Maria Stenkil; Jorsal, Anders;

    2012-01-01

    Placental growth factor is a vascular endothelial growth factor involved in angiogenesis, vascular inflammation and plaque formation. Soluble Fms-like tyrosine kinase 1 is a decoy receptor for placental growth factor, reducing its activity. The aim of this study is to evaluate the predictive valu...... of placental growth factor and soluble Fms-like tyrosine kinase 1 in relation to all-cause and cardiovascular mortality and decline in kidney function in Type 1 diabetes....

  10. Sesquiterpene dimmer (DSF-27) inhibits the release of neuroinflammatory mediators from microglia by targeting spleen tyrosine kinase (Syk) and Janus kinase 2 (Jak2): Two major non-receptor tyrosine signaling proteins involved in inflammatory events

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Ke-Wu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Wang, Shu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Department of Medicinal Chemistry and Pharmaceutical Analysis, Logistics College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Dong, Xin; Jiang, Yong; Jin, Hong-Wei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Tu, Peng-Fei, E-mail: pengfeitu@vip.163.com [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China)

    2014-03-15

    Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immunoregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways. - Highlights: • Sesquiterpene dimmer DSF-27 inhibits inflammatory mediators' production in microglia. • Syk-dependent Akt/NF-κB pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 signaling pathway is partly regulated by ERK and p38 MAPKs and PIAS3. • DSF-27 protects neurons against microglia-mediated neuroinflammatory injury.

  11. Tyrosine kinase inhibitors for epidermal growth factor receptor gene mutation-positive non-small cell lung cancers: an update for recent advances in therapeutics.

    Science.gov (United States)

    Chung, Clement

    2016-06-01

    The presence of activating gene mutations in the epidermal growth factor receptor of non-small cell lung cancer patients is predictive (improved progression-free survival and improved response rate) when treated with small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and afatinib. The two most common mutations that account for greater than 85% of all EGFR gene mutations are in-frame deletions in exon 19 (LREA deletions) and substitution in exon 21 (L858R). Exon 18 mutations occur much less frequently at about 4% of all EGFR gene mutations. Together, exon 19 deletion and exon 21 L858R gene substitution are present in about 10% of Caucasian patients and 20-40% of Asian patients with non-small cell lung cancer. T790M gene mutation at exon 20 is associated with acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Early studies showed that activating EGFR gene mutations are most common in patients with adenocarcinoma histology, women, never smokers and those of Asian ethnicity. A recent multi-center phase III trial suggested that frontline epidermal growth factor receptor tyrosine kinase inhibitor therapy with afatinib is associated with improved progression-free survival compared to chemotherapy regardless of race. Moreover, guidelines now suggest EGFR gene mutation testing should be conducted in all patients with lung adenocarcinoma or mixed lung cancers with an adenocarcinoma component, regardless of characteristics such as smoking status, gender or race. The success of targeted therapies in non-small cell lung cancer patients has changed the treatment paradigm in metastatic non-small cell lung cancer. However, despite a durable response of greater than a year, resistance to epidermal growth factor receptor tyrosine kinase inhibitors inevitably occurs. This mini-review describes the clinically relevant EGFR gene mutations and the efficacy/toxicity of small molecule epidermal growth factor receptor tyrosine kinase

  12. Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Galpha12/13.

    Science.gov (United States)

    Mao, J; Xie, W; Yuan, H; Simon, M I; Mano, H; Wu, D

    1998-10-01

    A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec non-receptor tyrosine kinase family. We found that Tec and Bmx activate serum response factor (SRF), in synergy with constitutively active alpha subunits of the G12 family of GTP-binding proteins, in transiently transfected NIH 3T3 cells. The SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the kinases are required for SRF activation. In addition, kinase-deficient mutants of Bmx are able to inhibit Galpha13- and Galpha12-induced SRF activation, and to suppress thrombin-induced SRF activation in cells lacking Galphaq/11, where thrombin's effect is mediated by G12/13 proteins. Moreover, expression of Galpha12 and Galpha13 stimulates autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in Galpha12/13-induced, Rho-mediated activation of SRF. Furthermore, Src, which was previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rho-independent pathways in 3T3 cells, as shown by the fact that C3 did not block Src-mediated SRF activation. However, the Rho-dependent pathway becomes significant when Tec is overexpressed.

  13. Dopamine D1-dependent trafficking of striatal N-methyl-D-aspartate glutamate receptors requires Fyn protein tyrosine kinase but not DARPP-32.

    Science.gov (United States)

    Dunah, Anthone W; Sirianni, Ana C; Fienberg, Allen A; Bastia, Elena; Schwarzschild, Michael A; Standaert, David G

    2004-01-01

    Interactions between dopaminergic and glutamatergic systems in the striatum are thought to underlie both the symptoms and adverse effects of treatment of Parkinson's disease. We have previously reported that activation of the dopamine D1 receptor triggers a rapid redistribution of striatal N-methyl-d-aspartate (NMDA) receptors between intracellular and postsynaptic sub-cellular compartments. To unravel the signaling pathways underlying this trafficking, we studied mice with targeted disruptions of either the gene that encodes the dopamine- and cAMP-regulated phosphoprotein (DARPP-32), a potent and selective inhibitor of protein phosphatase-1, or the protein tyrosine kinase Fyn. In striatal tissue from DARPP-32-depleted mice, basal tyrosine and serine phosphorylation of striatal NMDA receptor subunits NR1, NR2A, and NR2B was normal, and activation of dopamine D1 receptors with the agonist SKF-82958 [(+/-)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetra-hydro-1H-benzazepine] produced redistribution of NMDA receptors from vesicular compartments (P3 and LP2) to synaptosomal membranes (LP1). In the Fyn knockout mice, basal tyrosine phosphorylation of NR2A and NR2B was drastically reduced, whereas serine phosphorylation of these NMDA subunits was unchanged. In the Fyn knockout mice, the dopamine D1 receptor agonist failed to induce subcellular redistribution of NMDA receptors. In addition, Fyn-depleted mice lesioned with 6-hydroxydopamine also failed to exhibit l-DOPA-induced behavioral sensitization, but this may be caused, at least in part, by resistance of these mice to the neurotoxic lesion. These findings suggest a novel mechanism for the trafficking of striatal NMDA receptors by signaling pathways that are independent of DARPP-32 but require Fyn protein tyrosine kinase. Strategies that prevent NMDA receptor subcellular redistribution through inhibition of Fyn kinase may prove useful in the treatment of Parkinson's disease.

  14. Poorer prognosis in Taiwanese female ever smokers with stage IV lung adenocarcinoma who were readministered a tyrosine kinase inhibitor

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    Yang CJ

    2016-03-01

    Full Text Available Chih-Jen Yang,1–4 Ming-Ju Tsai,2 Jen-Yu Hung,2,4 Ying-Ming Tsai,1–3 Jui-Ying Lee,5 Shah-Hwa Chou,5,6 Ta-Chih Liu,7,8 Mei-Chiou Shen,9 Ming-Shyan Huang,2,4,10 Inn-Wen Chong2,6 1Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, 2Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 3Department of Internal Medicine, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 4Department of Internal Medicine, School of Medicine, College of Medicine, Kaohsiung Medical University, 5Division of Chest Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 6Department of Respiratory Care, College of Medicine, Kaohsiung Medical University, 7Division of Hematology and Oncology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 8Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, 9Department of Pharmacy, Kaohsiung Medical University Hospital, 10Division of Geriatric Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Background: Readministering a second epidermal growth factor receptor (EGFR tyrosine kinase inhibitor (TKI in patients with lung adenocarcinoma with acquired resistance to an initial EGFR TKI is a common treatment strategy. However, the prognostic factors for the second EGFR TKI are still uncertain.  Patients and methods: In this retrospective study, we enrolled patients with stage IV lung adenocarcinoma diagnosed between June 2009 and October 2013 at two university-affiliated hospitals in Taiwan. Basic characteristics including age, sex, smoking status, performance status, EGFR mutation status, tumor response, and progression-free survival (PFS of the second

  15. Coarse-grained molecular simulation of epidermal growth factor receptor protein tyrosine kinase multi-site self-phosphorylation.

    Directory of Open Access Journals (Sweden)

    John G Koland

    2014-01-01

    Full Text Available Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR, the intrinsic protein tyrosine kinase (PTK activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites in either of the two C-terminal (CT domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in

  16. Heightened cleavage of Axl receptor tyrosine kinase by ADAM metalloproteases may contribute to disease pathogenesis in SLE.

    Science.gov (United States)

    Orme, Jacob J; Du, Yong; Vanarsa, Kamala; Mayeux, Jessica; Li, Li; Mutwally, Azza; Arriens, Cristina; Min, Soyoun; Hutcheson, Jack; Davis, Laurie S; Chong, Benjamin F; Satterthwaite, Anne B; Wu, Tianfu; Mohan, Chandra

    2016-08-01

    Systemic lupus erythematosus (SLE) is characterized by antibody-mediated chronic inflammation in the kidney, lung, skin, and other organs to cause inflammation and damage. Several inflammatory pathways are dysregulated in SLE, and understanding these pathways may improve diagnosis and treatment. In one such pathway, Axl tyrosine kinase receptor responds to Gas6 ligand to block inflammation in leukocytes. A soluble form of the Axl receptor ectodomain (sAxl) is elevated in serum from patients with SLE and lupus-prone mice. We hypothesized that sAxl in SLE serum originates from the surface of leukocytes and that the loss of leukocyte Axl contributes to the disease. We determined that macrophages and B cells are a source of sAxl in SLE and in lupus-prone mice. Shedding of the Axl ectodomain from the leukocytes of lupus-prone mice is mediated by the matrix metalloproteases ADAM10 and TACE (ADAM17). Loss of Axl from lupus-prone macrophages renders them unresponsive to Gas6-induced anti-inflammatory signaling in vitro. This phenotype is rescued by combined ADAM10/TACE inhibition. Mice with Axl-deficient macrophages develop worse disease than controls when challenged with anti-glomerular basement membrane (anti-GBM) sera in an induced model of nephritis. ADAM10 and TACE also mediate human SLE PBMC Axl cleavage. Collectively, these studies indicate that increased metalloprotease-mediated cleavage of leukocyte Axl may contribute to end organ disease in lupus. They further suggest dual ADAM10/TACE inhibition as a potential therapeutic modality in SLE.

  17. Treatment compliance and severe adverse events limit the use of tyrosine kinase inhibitors in refractory thyroid cancer

    Directory of Open Access Journals (Sweden)

    Chrisoulidou A

    2015-09-01

    Full Text Available Alexandra Chrisoulidou, Stylianos Mandanas, Efterpi Margaritidou, Lemonia Mathiopoulou, Maria Boudina, Konstantinos Georgopoulos, Kalliopi Pazaitou-PanayiotouDepartment of Endocrinology, Theagenio Cancer Hospital, Thessaloniki, GreeceObjective: The aim of the present study was to assess patient compliance with tyrosine kinase inhibitor (TKI treatment used for refractory and progressive thyroid cancer, in addition to the efficacy and serious adverse events associated with these agents.Methods: We retrospectively analyzed data from adult patients with metastatic differentiated or medullary thyroid cancer unresponsive to conventional treatment and treated with TKIs. Patients received treatment until disease progression or onset of serious adverse events, or until they expressed an intention to stop treatment.Results: Twenty-four patients received TKIs. The median duration of treatment was four (range: 1–19 cycles. The most frequent adverse events were fatigue, nausea, diarrhea, hypertension, and stomatitis, and the most severe were nasal bleeding, diarrhea, heart failure, rhabdomyolysis, renal failure, QT prolongation, neutropenia, and severe fatigue. Dose reduction was required in eight patients, while five decided to terminate TKI therapy because adverse events impaired their everyday activities. During therapy, two patients showed a partial response and three showed stable disease. The lungs were the metastatic sites favoring a response to treatment.Conclusion: Patient selection and meticulous pretreatment education are necessary in order to ensure adherence with TKI therapy. If adverse events appear, dose reduction or temporary treatment interruption may be offered because some adverse events resolve with continuation of treatment. In the event of serious adverse events, treatment discontinuation is necessary. Keywords: medullary thyroid carcinoma, differentiated thyroid cancer, TKIs, sorafenib, sunitinib, vandetanib

  18. A new solvate of afatinib, a specific inhibitor of the ErbB family of tyrosine kinases

    Directory of Open Access Journals (Sweden)

    Matthias Zeller

    2017-03-01

    Full Text Available Afatinib (systematic name: N-{4-(3-chloro-4-fluoroanilino-7-[(tetrahydrofuran-3-yloxy]quinazolin-6-yl}-4-(dimethylaminobut-2-enamide, is a specific inhibitor of the ErbB family of tyrosine kinases. The free base form crystallizes from acetonitrile as a mixed water–acetonitrile solvent, C24H25ClFN5O3·0.25C2H3N·2H2O. It crystallizes with two independent molecules (A and B in the asymmetric unit of the chiral space group P4212, but exhibits close to perfect pseudo-inversion symmetry, emulating P4/ncc that relates the two molecules to each other. Exact inversion symmetry is however broken by swapping of oxygen and CH2 moieties of the outer tetrahydrofuranyl substituents of the two independent molecules. This can, in turn, be traced back to C—H...N and C—H...O interactions of the acetonitrile solvent molecules with the tetrahydrofuran oxygen and CH2 units. In the crystal, neighboring molecules are connected via N—H...O hydrogen bonds between the secondary amine and the amide keto O atom. Additional hydrogen bonds are formed through the water solvent molecules, which are engaged in O—H...O and O—H...N hydrogen bonds connecting to the dimethylamino N atom, the amide keto O atom, and one of the quinazoline N atoms of a neighboring molecule, leading to an intricate three-dimensional hydrogen-bonded superstructure. There are two types of channels stretching along the direction of the c axis; one along the fourfold rotational axis, occupied by acetonitrile solvent molecules situated on that axis, and parallel channels which are not occupied by any solvent.

  19. In vitro hepatic metabolism of cediranib, a potent vascular endothelial growth factor tyrosine kinase inhibitor: interspecies comparison and human enzymology.

    Science.gov (United States)

    Schulz-Utermoehl, Timothy; Spear, Michael; Pollard, Christopher R J; Pattison, Christine; Rollison, Helen; Sarda, Sunil; Ward, Michelle; Bushby, Nick; Jordan, Angela; Harrison, Mike

    2010-10-01

    The in vitro metabolism of cediranib (4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-[3-(1-pyrrolidinyl)propoxy]quinazoline), a vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor (TKI) of all three VEGF receptors in late-stage development for the treatment of colorectal cancer and recurrent glioblastoma was investigated in hepatic proteins from preclinical species and humans using radiolabeled material. In human hepatocyte cultures, oxidative and conjugative metabolic pathways were identified, with pyrrolidine N(+)-glucuronidation being the major route. The primary oxidative pathways were di-and trioxidations and pyrrolidine N-oxidation. All metabolites with the exception of the N(+)-glucuronide metabolite were observed in rat and cynomolgus monkey hepatocyte preparations. Additional metabolism studies in liver microsomes from these or other preclinical species (CD-1 mouse, Han Wistar rat, Dunkin Hartley guinea pig, Göttingen mini-pig, New Zealand White rabbit, beagle dog, and cynomolgus and rhesus monkey) indicated that the N(+)-glucuronide metabolite was not formed in these additional species. Incubations with recombinant flavin-containing monooxygenase (FMO) and UDP-glucuronosyltransferase (UGT) enzymes and inhibition studies using the nonselective cytochrome P450 (P450) chemical inhibitor 1-aminobenzotriazole in human hepatocytes indicated that FMO1 and FMO3 contributed to cediranib N-oxidation, whereas UGT1A4 had a major role in cediranib N(+)-glucuronidation. P450 enzymes had only a minor role in the metabolism of cediranib. In conclusion, species differences in the formation of the N(+)-glucuronide metabolite of cediranib were observed. All other metabolites of cediranib found in humans were also detected in rat and cynomolgus monkey. Non-P450 enzymes are predominantly involved in the metabolism of cediranib, and this suggests that clinical drug interactions involving other coadministered drugs are unlikely.

  20. EphA4 receptor tyrosine kinase is a modulator of onset and disease severity of experimental autoimmune encephalomyelitis (EAE.

    Directory of Open Access Journals (Sweden)

    Kathryn M Munro

    Full Text Available The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE, axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections. EphA4 knockout mice exhibited a markedly less severe clinical course than wildtype mice, with a lower maximum disease grade and a slightly later onset of clinical symptoms. Numbers of infiltrating T cells and macrophages, the number and size of the lesions, and the extent of astrocytic gliosis were similar in both genotypes; however, EphA4 knockout mice appeared to have decreased axonal pathology. Blocking of EphA4 in wildtype mice by administration of soluble EphA4 (EphA4-Fc as a decoy receptor following induction of EAE produced a delay in onset of clinical symptoms; however, most mice had clinical symptoms of similar severity by 22 days, indicating that EphA4 blocking treatment slowed early EAE disease evolution. Again there were no apparent differences in histopathology. To determine whether the role of EphA4 in modulating EAE was CNS mediated or due to an altered immune response, MOG primed T cells from wildtype and EphA4 knockout mice were passively transferred into naive recipient mice and both were shown to induce disease of equivalent severity. These results are consistent with a non-inflammatory, CNS specific, deleterious effect of EphA4 during neuroinflammation that results in axonal pathology.

  1. The anti-esophageal cancer cell activity by a novel tyrosine/phosphoinositide kinase inhibitor PP121

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Yi; Zhou, Yajuan [Department of Radiation and Medical Oncology, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Department of Radiation Oncology, Hubei Cancer Hospital, Wuhan 430071 (China); Cheng, Long [Department of Interventional Radiology, the Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215001 (China); Hu, Desheng; Zhou, Xiaoyi; Wang, Zhaohua [Department of Radiation Oncology, Hubei Cancer Hospital, Wuhan 430071 (China); Xie, Conghua, E-mail: chxie_65@hotmail.com [Department of Radiation and Medical Oncology, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Zhou, Fuxiang, E-mail: ZhouFuxiangwuhan@126.com [Department of Radiation and Medical Oncology, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China)

    2015-09-11

    Here we explored the potential effect of PP121, a novel dual inhibitor of tyrosine and phosphoinositide kinases, against human esophageal cancer cells. We showed that PP121 exerted potent cytotoxic effect in primary (patient-derived) and established (Eca-109, TE-1 and TE-3 lines) esophageal cancer cells, possibly through activating caspase-3-dependnent apoptosis. PP121 was, however, non-cytotoxic to the normal human esophageal epithelial cells (EECs). At the molecular level, we showed that PP121 blocked Akt-mTOR (mammalian target of rapamycin) activation in esophageal cancer cells, which was restored by introducing a constitutively-active Akt (CA-Akt). Yet, CA-Akt only partly inhibited cytotoxicity by PP121 in Eca-109 cells. Importantly, we showed that PP121 inhibited nuclear factor kappa B (NFκB) signaling activation in esophageal cancer cells, which appeared independent of Akt-mTOR blockage. In vivo, oral administration of PP121 remarkably inhibited Eca-109 xenograft growth in nude mice, and significantly improved mice survival. Further, the immunohistochemistry (IHC) and Western blot assays analyzing xenografted tumors showed that PP121 inhibited Akt-mTOR and NFκB activations in vivo. Together, we demonstrate that PP121 potently inhibits esophageal cancer cells in vitro and in vivo, possibly through concurrently inhibiting Akt-mTOR and NFκB signalings. - Highlights: • PP121 is cytotoxic against primary and established esophageal cancer cells. • PP121 induces caspase-3-dependnent apoptosis in esophageal cancer cells. • PP121 blocks Akt-mTOR activation in esophageal cancer cells. • PP121 inhibits NFκB activation, independent of Akt-mTOR blockage. • PP121 inhibits Eca-109 xenograft growth and Akt-mTOR/NFκB activation in vivo.

  2. Expression of Brain-derived Neurotrophic Factor and Tyrosine Kinase B in Cerebellum of Poststroke Depression Rat Model

    Institute of Scientific and Technical Information of China (English)

    Yun Li; Chun Peng; Xu Guo; Jun-Jie You; Harishankar Prasad Yadav

    2015-01-01

    Background:The pathophysiology of poststroke depression (PSD) remains elusive because of its proposed multifactorial nature.Accumulating evidence suggests that brain-derived neurotrophic factor (BDNF) plays a key role in the pathophysiology of depression and PSD.And the cerebellar dysfunction may be important in the etiology of depression;it is not clear whether it also has a major effect on the risk of PSD.This study aimed to explore the expression of BDNF and high-affinity receptors tyrosine kinase B (TrkB) in the cerebellum of rats with PSD.Methods:The rat models with focal cerebral ischemic were made using a thread embolization method.PSD rat models were established with comprehensive separate breeding and unpredicted chronic mild stress (UCMS) on this basis.A normal control group,depression group,and a stroke group were used to compare with the PSD group.Thirteen rats were used in each group.Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) for detecting the expression of BDNF and TrkB protein and mRNA in the cerebellum were used at the 29th day following the UCMS.Results:Compared with the normal control group and the stroke group,the number ofBDNF immunoreactive (IR) positive neurons was less in the PSD group (P < 0.05).Furthermore,the number ofTrkB IR positive cells was significantly less in the PSD group than that in the normal control group (P < 0.05).The gene expression of BDNF and TrkB in the cerebellum of PSD rats also decreased compared to the normal control group (P < 0.05).Conclusions:These findings suggested a possible association between expression of BDNF and TrkB in the cerebellum and the pathogenesis of PSD.

  3. Expression of Brain-derived Neurotrophic Factor and Tyrosine Kinase B in Cerebellum of Poststroke Depression Rat Model

    Directory of Open Access Journals (Sweden)

    Yun Li

    2015-01-01

    Full Text Available Background: The pathophysiology of poststroke depression (PSD remains elusive because of its proposed multifactorial nature. Accumulating evidence suggests that brain-derived neurotrophic factor (BDNF plays a key role in the pathophysiology of depression and PSD. And the cerebellar dysfunction may be important in the etiology of depression; it is not clear whether it also has a major effect on the risk of PSD. This study aimed to explore the expression of BDNF and high-affinity receptors tyrosine kinase B (TrkB in the cerebellum of rats with PSD. Methods: The rat models with focal cerebral ischemic were made using a thread embolization method. PSD rat models were established with comprehensive separate breeding and unpredicted chronic mild stress (UCMS on this basis. A normal control group, depression group, and a stroke group were used to compare with the PSD group. Thirteen rats were used in each group. Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR for detecting the expression of BDNF and TrkB protein and mRNA in the cerebellum were used at the 29 th day following the UCMS. Results: Compared with the normal control group and the stroke group, the number of BDNF immunoreactive (IR positive neurons was less in the PSD group (P < 0.05. Furthermore, the number of TrkB IR positive cells was significantly less in the PSD group than that in the normal control group (P < 0.05. The gene expression of BDNF and TrkB in the cerebellum of PSD rats also decreased compared to the normal control group (P < 0.05. Conclusions: These findings suggested a possible association between expression of BDNF and TrkB in the cerebellum and the pathogenesis of PSD.

  4. A fresh perspective on comparing the FDA and the CHMP/EMA: approval of antineoplastic tyrosine kinase inhibitors.

    Science.gov (United States)

    Shah, Rashmi R; Roberts, Samantha A; Shah, Devron R

    2013-09-01

    We compared and determined the reasons for any differences in the review and approval times of tyrosine kinase inhibitors (TKIs) by the US Food and Drug Administration (FDA) and the European EMA/CHMP. Applications for these novel cancer drugs were submitted to them within a mean of 31.2 days of each other, providing a fair basis for comparison. The FDA had granted priority review to 12 TKIs but the EMA/CHMP did not grant the equivalent accelerated assessment to any. The FDA granted accelerated approvals to six (38%) and CHMP granted (the equivalent) conditional approvals to four (29%) of these agents. On average, the review and approval times were 205.3 days in the US compared with 409.6 days in the European Union (EU). The active review times, however, were comparable (225.4 days in the EU and 205.3 days in the US). Since oncology drug development lasts about 7 years, the 20 days difference in review times between the two agencies is inconsequential. Clock stops during review and the time required to issue an approval had added the extra 184.2 days to review time in the EU. We suggest possible solutions to expedite the EU review and approval processes. However, post-marketing emergence of adverse efficacy and safety data on gefitinib and lapatinib, respectively, indicate potential risks of expedited approvals. We challenge the widely prevalent myth that early approval translates into early access or beneficial impact on public health. Both the agencies collaborate closely but conduct independent assessments and make decisions based on distinct legislation, procedures, precedents and societal expectations.

  5. Radiosensitivity of tumor cell lines after pretreatment with the EGFR tyrosine kinase inhibitor ZD1839 (Iressa {sup registered})

    Energy Technology Data Exchange (ETDEWEB)

    Burdak-Rothkamm, S. [Dept. of Radiotherapy, Saarland Univ. Hospital, Homburg/Saar (Germany); Gray Cancer Inst., Northwood, Middlesex (United Kingdom); Ruebe, C.E.; Nguyen, T.P.; Ludwig, D.; Ruebe, C. [Dept. of Radiotherapy, Saarland Univ. Hospital, Homburg/Saar (Germany); Feldmann, K. [AstraZeneca GmbH, Wedel (Germany); Wiegel, T. [Dept. of Radiotherapy, Univ. Hospital Benjamin Franklin, Berlin (Germany)

    2005-03-01

    Background and purpose: the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 (Iressa registered) reduces survival and augments radiation response of certain tumor cells. The aim of this study was to identify cellular events that are associated with the modulation of radiosensitivity by ZD1839. Material and methods: three tumor cell lines (A549, H596, FaDu) were exposed to ionizing radiation, treatment with ZD1839, and combined treatment. Clonogenic cell survival was determined by colony assays, EGFR and transforming growth factor-(TGF-){alpha} expression by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), cell cycle distribution and apoptosis by flow cytometry. Results: in A549 and H596 cells ZD1839 had little effect on clonogenic growth, but survival curves revealed a radiosensitizing effect of 5 {mu}M ZD1839 on A549 cells. Both cell lines expressed moderate amounts of EGFR mRNA and very low levels of TGF-{alpha} mRNA. FaDu cells expressed relatively high amounts of EGFR and TGF-{alpha} transcripts and showed marked inhibition of clonogenic growth, reduction of S-phase cells, and induction of apoptosis after treatment with 1 {mu}M ZD1839 and combined treatment. Surprisingly, the subpopulation of FaDu cells surviving ZD1839 pretreatment was more radioresistant. Exposure to ZD1839 caused a decrease in EGFR mRNA expression in A549 cells, no change in H596, and even an increase in FaDu cells. Conclusion: the sensitivity to ZD1839 correlated with the EGFR expression level, an inhibition of cell proliferation, and induction of apoptosis in the cell lines analyzed. A radiosensitizing effect of ZD1839 was associated with downregulation of EGFR mRNA expression. (orig.)

  6. Characterization and response of newly developed high-grade glioma cultures to the tyrosine kinase inhibitors, erlotinib, gefitinib and imatinib

    Energy Technology Data Exchange (ETDEWEB)

    Kinsella, Paula, E-mail: paula.kinsella@dcu.ie [National Institute for Cellular Biotechnology, Dublin City University, Dublin 9 (Ireland); Howley, Rachel, E-mail: rhowley@rcsi.ie [Department of Neuropathology, Beaumont Hospital, Dublin 9 (Ireland); Doolan, Padraig, E-mail: padraig.doolan@dcu.ie [National Institute for Cellular Biotechnology, Dublin City University, Dublin 9 (Ireland); Clarke, Colin, E-mail: colin.clarke@dcu.ie [National Institute for Cellular Biotechnology, Dublin City University, Dublin 9 (Ireland); Madden, Stephen F., E-mail: maddens@dcu.ie [National Institute for Cellular Biotechnology, Dublin City University, Dublin 9 (Ireland); Clynes, Martin, E-mail: Martin.Clynes@dcu.ie [National Institute for Cellular Biotechnology, Dublin City University, Dublin 9 (Ireland); Farrell, Michael, E-mail: michaelfarrell@beaumont.ie [Department of Neuropathology, Beaumont Hospital, Dublin 9 (Ireland); Amberger-Murphy, Verena, E-mail: Verena.Murphy@icorg.ie [National Institute for Cellular Biotechnology, Dublin City University, Dublin 9 (Ireland); All Ireland Co-operative, Oncology Research Group, 60 Fitzwilliam Square, Dublin 2 (Ireland)

    2012-03-10

    High-grade gliomas (HGG), are the most common aggressive brain tumours in adults. Inhibitors targeting growth factor signalling pathways in glioma have shown a low clinical response rate. To accurately evaluate response to targeted therapies further in vitro studies are necessary. Growth factor pathway expression using epidermal growth factor receptor (EGFR), mutant EGFR (EGFRvIII), platelet derived growth factor receptor (PDGFR), C-Kit and C-Abl together with phosphatase and tensin homolog (PTEN) expression and downstream activation of AKT and phosphorylated ribosomal protein S6 (P70S6K) was analysed in 26 primary glioma cultures treated with the tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib and imatinib. Response to TKIs was assessed using 50% inhibitory concentrations (IC{sub 50}). Response for each culture was compared with the EGFR/PDGFR immunocytochemical pathway profile using hierarchical cluster analysis (HCA) and principal component analysis (PCA). Erlotinib response was not strongly associated with high expression of the growth factor pathway components. PTEN expression did not correlate with response to any of the three TKIs. Increased EGFR expression was associated with gefitinib response; increased PDGFR-{alpha} expression was associated with imatinib response. The results of this in vitro study suggest gefitinib and imatinib may have therapeutic potential in HGG tumours with a corresponding growth factor receptor expression profile. -- Highlights: Black-Right-Pointing-Pointer Non-responders had low EGFR expression, high PDGFR-{beta}, and a low proliferation rate. Black-Right-Pointing-Pointer PTEN is not indicative of response to a TKI. Black-Right-Pointing-Pointer Erlotinib response was not associated with expression of the proteins examined. Black-Right-Pointing-Pointer Imatinib-response correlated with expression of PDGFR-{alpha}. Black-Right-Pointing-Pointer Gefitinib response correlated with increased expression of EGFR.

  7. Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Takayuki [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Murata, Kazuko, E-mail: murata-k@iwakimu.ac.jp [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Murata, Ryo [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Sun, Shu-lan [Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Saito, Yutaro; Yamaga, Shuhei [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Tanaka, Nobuyuki; Tamai, Keiichi [Division of Immunology, Miyagi Cancer Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293 (Japan); Moriya, Kunihiko [Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Kasai, Noriyuki [Institute for Animal Experimentation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Sugamura, Kazuo [Division of Immunology, Miyagi Cancer Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293 (Japan); Ishii, Naoto [Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan)

    2014-01-10

    Highlights: •ESCRT-0 protein regulates the development of peripheral B-cells. •BCR expression on cell surface should be controlled by the endosomal-sorting system. •Hrs plays important roles in responsiveness to Ag stimulation in B lymphocytes. -- Abstract: Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs{sup flox/flox};mb1{sup cre/+}:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

  8. Impact of TP53 Mutations on Outcome in EGFR-Mutated Patients Treated with First-Line Tyrosine Kinase Inhibitors.

    Science.gov (United States)

    Canale, Matteo; Petracci, Elisabetta; Delmonte, Angelo; Chiadini, Elisa; Dazzi, Claudio; Papi, Maximilian; Capelli, Laura; Casanova, Claudia; De Luigi, Nicoletta; Mariotti, Marita; Gamboni, Alessandro; Chiari, Rita; Bennati, Chiara; Calistri, Daniele; Ludovini, Vienna; Crinò, Lucio; Amadori, Dino; Ulivi, Paola

    2016-10-25

    Purpose: To analyze the impact of TP53 mutations on response to first-line tyrosine kinase inhibitors (TKI) in patients with EGFR-mutated non-small cell lung cancer (NSCLC).Experimental Design: 136 EGFR-mutated NSCLC patients receiving first-line TKIs were analyzed. TP53 mutations were evaluated in 123 patients in relation to disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS), and overall survival (OS).Results:TP53 mutations were observed in 37 (30.1%), 10 (27.0%), 6 (16.2%), 9 (24.3%), and 12 (32.4%) patients in exons 5, 6, 7, and 8, respectively. DCR was 70% in TP53-mutated patients compared with 88% in TP53-wild type (wt) patients [relative risk, RR, of disease progression: 3.17 (95% CI, 1.21-8.48), P = 0.019]. In particular, a 42% DCR was observed in patients with TP53 exon 8 mutation versus 87% in exon 8 wt patients [RR of disease progression 9.6 (2.71-36.63), P TP53 exon 8 mutations compared with others (4.2 vs. 12.5, P = 0.058, and 16.2 vs. 32.3, P = 0.114, respectively); these differences became significant in the subgroup with EGFR exon 19 deletion (4.2 vs. 16.8, P TP53 mutations, especially exon 8 mutations, reduce responsiveness to TKIs and worsen prognosis in EGFR-mutated NSCLC patients, mainly those carrying exon 19 deletions. Clin Cancer Res; 1-8. ©2016 AACR.

  9. Modulation of liver canalicular transport processes by the tyrosine-kinase inhibitor genistein: implications of genistein metabolism in the rat.

    Science.gov (United States)

    Jäger, W; Winter, O; Halper, B; Salamon, A; Sartori, M; Gajdzik, L; Hamilton, G; Theyer, G; Graf, J; Thalhammer, T

    1997-12-01

    Rat liver cells express the multispecific organic anion transporter (cmoat, cmrp, mrp2) and P-glycoprotein (Pgp) in their canalicular membranes, proteins that are homologous to the multidrug-resistance related protein (MRP) and multidrug resistance (MDR) gene products in multidrug resistant tumor cells. We tested whether genistein, a modulator of drug resistance in tumor cells, affects biliary secretion of substrates of canalicular multispecific organic anion transporter (cmoat) (glucuronides of bilirubin and rhodamine, glutathione conjugate of bromsulphthalein) and of P-glycoprotein (Pgp) (rhodamine), respectively. Using the isolated perfused rat liver of control Wistar rats (TR+) and of a mutant strain (TR-) that expresses Pgp but not cmoat, we show that genistein effectively inhibits the secretion of anionic substrates of cmoat in Wistar rats but stimulates secretion of cationic rhodamine in TR- rats. Genistein is subject to glucuronidation and sulfatation and secretion of genistein and its metabolites stimulates bile flow in Wistar rats, but secretion is nearly absent in TR- rats. Because genistein and its metabolites are substrates for cmoat, inhibition of anion secretion by genistein is partially explained by competition for this transporter. Genistein is also a substrate of uridindiphosphate (UDP)-glucuronyltransferase isoenzyme(s). Inhibition of glucuronidation reduces the availability of bilirubin and rhodamine glucuronates for transport via cmoat, but unconjugated cationic rhodamine becomes available for transport via Pgp at an increased cellular concentration. Daidzein, a genistein analogue with no effect on protein tyrosine kinase (PTK) shows Similar effects on secretion of organic anions and cations supporting the conclusion that genistein affects transport in liver mainly through competition with other substrates at the sites of glucuronidation and transport via cmoat.

  10. Ultrasensitive electrochemical detection of protein tyrosine kinase-7 by gold nanoparticles and methylene blue assisted signal amplification.

    Science.gov (United States)

    Miao, Xiangmin; Li, Zongbing; Zhu, Aihua; Feng, Zhaozhong; Tian, Jun; Peng, Xue

    2016-09-15

    We present here an ultrasensitive and simple strategy for protein tyrosine kinase-7 (PTK7) detection based on the recognition-induced structure change of sgc8 aptamer, and the signal change of methylene blue (MB) that interacted with sandwiched DNA complex. To construct such a sensor, an homogeneous nano-surface was formed firstly on the glass carbon electrode (GCE) by using negatively charged Nafion (Nf) as the inner layer and positively charged gold nanoparticles ((+)AuNPs) as the outer layer, followed by the immobilization of sgc8 aptamer based on Au-S bond. In the presence of helper probe (HP), sandwiched DNA complex was formed between the sgc8 aptamer and the DNA modified gold nanoparticle probe (DNA-AuNPs). Then, a strong current signal was produced due to the capture of abundant MB molecules by both the sandwiched DNA complex and the multiple DNAs that modified on AuNPs surface. However, the specific binding of sgc8 aptamer with PNK7 would trigger a structure transition of it, and directly prevented the following formation of sandwiched structure and the capture of MB. Thus, PTK7 detection could be realized based on monitoring the signal reduction of MB upon incubation of sgc8 aptamer with PTK7. Under optimal conditions, a low detection limit of 372 fM was obtained for PNK7 detection. Due to the employment of sgc8 aptamer, the proposed biosensor exhibited high selectivity to PNK7. Moreover, satisfactory results were obtained when the proposed method was applied for PNK7 detection in cellular debris.

  11. Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Keisuke Tabara

    Full Text Available Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs. However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2 or BIBW2992 (pan-TKI of EGFR family proteins. Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.

  12. CBL is frequently altered in lung cancers: its relationship to mutations in MET and EGFR tyrosine kinases.

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    Yi-Hung Carol Tan

    Full Text Available BACKGROUND: Non-small cell lung cancer (NSCLC is a heterogeneous group of disorders with a number of genetic and proteomic alterations. c-CBL is an E3 ubiquitin ligase and adaptor molecule important in normal homeostasis and cancer. We determined the genetic variations of c-CBL, relationship to receptor tyrosine kinases (EGFR and MET, and functionality in NSCLC. METHODS AND FINDINGS: Using archival formalin-fixed paraffin embedded (FFPE extracted genomic DNA, we show that c-CBL mutations occur in somatic fashion for lung cancers. c-CBL mutations were not mutually exclusive of MET or EGFR mutations; however they were independent of p53 and KRAS mutations. In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH for the c-CBL locus (22%, n = 8/37 and none of these samples revealed any mutation in the remaining copy of c-CBL. The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples. Using select c-CBL somatic mutations such as S80N/H94Y, Q249E and W802* (obtained from Caucasian, Taiwanese and African-American samples, respectively transfected in NSCLC cell lines, there was increased cell viability and cell motility. CONCLUSIONS: Taking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis.

  13. CD66-mediated phagocytosis of Opa52 Neisseria gonorrhoeae requires a Src-like tyrosine kinase- and Rac1-dependent signalling pathway.

    Science.gov (United States)

    Hauck, C R; Meyer, T F; Lang, F; Gulbins, E

    1998-01-15

    The interaction of Neisseria gonorrhoeae with human phagocytes is a hallmark of gonococcal infections. Recently, CD66 molecules have been characterized as receptors for Opa52-expressing gonococci on human neutrophils. Here we show that Opa52-expressing gonococci or Escherichia coli or F(ab) fragments directed against CD66, respectively, activate a signalling cascade from CD66 via Src-like protein tyrosine kinases, Rac1 and PAK to Jun-N-terminal kinase. The induced signal is distinct from Fcgamma-receptor-mediated signalling and is specific for Opa52, since piliated Opa- gonococci, commensal Neisseria cinerea or E.coli do not stimulate this signalling pathway. Inhibition of Src-like kinases or Rac1 prevents the uptake of Opa52 bacteria, demonstrating the crucial role of this signalling cascade for the opsonin-independent, Opa52/CD66-mediated phagocytosis of pathogenic Neisseria.

  14. Fulvestrant-induced cell death and proteasomal degradation of estrogen receptor α protein in MCF-7 cells require the CSK c-Src tyrosine kinase.

    Directory of Open Access Journals (Sweden)

    Wei-Lan Yeh

    Full Text Available Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD. In contrast to the Selective Estrogen Receptor Modulators (SERMs such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK, a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.

  15. Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes

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    Klopfleisch Robert

    2012-06-01

    Full Text Available Abstract Background Canine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumour type. However, little is known on the downstream target genes of this signaling pathway and molecular changes after inhibition. Results Transcriptome analysis of the canine mast cell tumour cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes or 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signaling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1. Conclusions Transcriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated, which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect.

  16. Inhibition of Src tyrosine kinase activity by squamosamide derivative FLZ attenuates neuroinflammation in both in vivo and in vitro Parkinson's disease models.

    Science.gov (United States)

    Tai, Wenjiao; Ye, Xuan; Bao, Xiuqi; Zhao, Baozhong; Wang, Xiaoliang; Zhang, Dan

    2013-12-01

    The participation of neuroinflammation in the pathogenesis of Parkinson's disease (PD) has long been validated. Excessive activated microglia release a large number of pro-inflammatory factors, damage surrounding neurons and eventually induce neurodegeneration. Inhibition of microglial over-activation might be a promising strategy for PD treatment. FLZ (formulated as: N-(2-(4-hydroxy-phenyl)-ethyl)-2-(2, 5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide, the code name: FLZ), a natural squamosamide derivative from a Chinese herb, has been shown to inhibit over-activated microglia and protect dopaminergic neurons in previous studies, but the mechanism remains unclear. In the present study, we further investigated the mechanism in lipopolysaccharide (LPS)-induced in vivo and in vitro PD models. FLZ treatment significantly improved the motor dysfunction of PD model rats induced by intra-nigral injection of LPS and this beneficial effect of FLZ attributed to the inhibition of microglial over-activation and the protection on dopaminergic neurons in the substantia nigra (SN). In vitro mechanistic study revealed that the inhibitive effect of FLZ on microglia was mediated by suppressing Src kinase related inflammatory signaling pathway activation and subsequent NF-κBp65 nuclear translocation, inhibiting nitric oxide (NO) and reactive oxygen species (ROS) production, decreasing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. In conclusion, the present study supports that FLZ exerts neuroprotection against LPS-induced dopaminergic neurodegeneration through its anti-inflammatory effect, which is mediated by suppressing Src tyrosine kinase and the downstream inflammatory signaling pathway. Furthermore, this study defines a critical role of Src tyrosine kinase in neuroinflammation, and suggests that particular tyrosine kinase inhibition may be a potential anti-inflammatory approach for PD treatment.

  17. A multiscale modeling approach to investigate molecular mechanisms of pseudokinase activation and drug resistance in the HER3/ErbB3 receptor tyrosine kinase signaling network.

    Science.gov (United States)

    Telesco, Shannon E; Shih, Andrew J; Jia, Fei; Radhakrishnan, Ravi

    2011-06-01

    Multiscale modeling provides a powerful and quantitative platform for investigating the complexity inherent in intracellular signaling pathways and rationalizing the effects of molecular perturbations on downstream signaling events and ultimately, on the cell phenotype. Here we describe the application of a multiscale modeling scheme to the HER3/ErbB3 receptor tyrosine kinase (RTK) signaling network, which regulates critical cellular processes including proliferation, migration and differentiation. The HER3 kinase is a topic of current interest and investigation, as it has been implicated in mechanisms of resistance to tyrosine kinase inhibition (TKI) of EGFR and HER2 in the treatment of many human malignancies. Moreover, the commonly regarded status of HER3 as a catalytically inactive 'pseudokinase' has recently been challenged by our previous study, which demonstrated robust residual kinase activity for HER3. Through our multiscale model, we investigate the most significant molecular interactions that contribute to potential mechanisms of HER3 activity and the physiological relevance of this activity to mechanisms of drug resistance in an ErbB-driven tumor cell in silico. The results of our molecular-scale simulations support the characterization of HER3 as a weakly active kinase that, in contrast to its fully-active ErbB family members, depends upon a unique hydrophobic interface to coordinate the alignment of specific catalytic residues required for its activity. Translating our molecular simulation results of the uniquely active behavior of the HER3 kinase into a physiologically relevant environment, our HER3 signaling model demonstrates that even a weak level of HER3 activity may be sufficient to induce AKT signaling and TKI resistance in the context of an ErbB signaling-dependent tumor cell, and therefore therapeutic targeting of HER3 may represent a superior treatment strategy for specific ErbB-driven cancers.

  18. In situ quantification of HER2–protein tyrosine kinase 6 (PTK6) protein–protein complexes in paraffin sections from breast cancer tissues

    Science.gov (United States)

    Aubele, M; Spears, M; Ludyga, N; Braselmann, H; Feuchtinger, A; Taylor, K J; Lindner, K; Auer, G; Stering, K; Höfler, H; Schmitt, M; Bartlett, J M S

    2010-01-01

    Background: Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression of PTK6 in tumours is linked with the expression of HER2. Method and results: In this study, we used the proximity ligation assay (PLA) technique on formalin-fixed paraffin sections from eighty invasive breast carcinoma tissue specimens to locate PTK6–HER2 protein–protein complexes. Proximity ligation assay signals from protein complexes were assessed quantitatively, and expression levels showed a statistically significant association with tumour size (P=0.015) and course of the cancer disease (P=0.012). Conclusion: Protein tyrosine kinase 6 forms protein complexes with HER2 in primary breast cancer tissues, which can be visualised by use of the PLA technique. Human epidermal growth factor receptor 2–PTK6 complexes are of prognostic relevance. PMID:20700126

  19. Compensatory upregulation of tyrosine kinase Etk/BMX in response to androgen deprivation promotes castration-resistant growth of prostate cancer cells.

    Science.gov (United States)

    Dai, Bojie; Chen, Hege; Guo, Shengjie; Yang, Xi; Linn, Douglas E; Sun, Feng; Li, Wei; Guo, Zhiyong; Xu, Kexin; Kim, Oekyung; Kong, Xiangtian; Melamed, Jonathan; Qiu, Shaopeng; Chen, Hegang; Qiu, Yun

    2010-07-01

    We previously showed that targeted expression of non-receptor tyrosine kinase Etk/BMX in mouse prostate induces prostate intraepithelial neoplasia, implying a possible causal role of Etk in prostate cancer development and progression. Here, we report that Etk is upregulated in both human and mouse prostates in response to androgen ablation. Etk expression seems to be differentially regulated by androgen and interleukin 6 (IL-6), which is possibly mediated by the androgen receptor (AR) in prostate cancer cells. Our immunohistochemical analysis of tissue microarrays containing 112 human prostate tumor samples revealed that Etk expression is elevated in hormone-resistant prostate cancer and positively correlated with tyrosine phosphorylation of AR (Pearson correlation coefficient rho = 0.71, P < 0.0001). AR tyrosine phosphorylation is increased in Etk-overexpressing cells, suggesting that Etk may be another tyrosine kinase, in addition to Src and Ack-1, which can phosphorylate AR. We also showed that Etk can directly interact with AR through its Src homology 2 domain, and such interaction may prevent the association of AR with Mdm2, leading to stabilization of AR under androgen-depleted conditions. Overexpression of Etk in androgen-sensitive LNCaP cells promotes tumor growth while knocking down Etk expression in hormone-insensitive prostate cancer cells by a specific shRNA that inhibits tumor growth under androgen-depleted conditions. Taken together, our data suggest that Etk may be a component of the adaptive compensatory mechanism activated by androgen ablation in prostate and may play a role in hormone resistance, at least in part, through direct modulation of the AR signaling pathway.

  20. A conditional form of Bruton's tyrosine kinase is sufficient to activate multiple downstream signaling pathways via PLC Gamma 2 in B cells

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    Witte Owen N

    2001-06-01

    Full Text Available Abstract Background Bruton's tyrosine kinase (Btk is essential for B cell development and function. Mutations of Btk elicit X-linked agammaglobulinemia in humans and X-linked immunodeficiency in the mouse. Btk has been proposed to participate in B cell antigen receptor-induced signaling events leading to activation of phospholipase C-γ2 (PLCγ2 and calcium mobilization. However it is unclear whether Btk activation is alone sufficient for these signaling events, and whether Btk can activate additional pathways that do not involve PLCγ2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCγ2-deficient DT40 B cell line. Results Activation of Btk:ER was sufficient to induce multiple B cell signaling pathways in PLCγ2-sufficient DT40 cells. These included tyrosine phosphorylation of PLCγ2, mobilization of intracellular calcium, activation of extracellular signal-regulated kinase (ERK and c-Jun NH2-terminal kinase (JNK mitogen-activated protein kinase (MAPK pathways, and apoptosis. In DT40 B cells deficient for PLCγ2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis. Conclusions These data suggest that Btk:ER regulates downstream signaling pathways primarily via PLCγ2 in B cells. While it is not known whether activated Btk:ER precisely mimics activated Btk, this conditional system will likely facilitate the dissection of the role of Btk and its family members in a variety of biological processes in many different cell types.

  1. SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Buchanan, Sean G.; Hendle, Jorg; Lee, Patrick S.; Smith, Christopher R.; Bounaud, Pierre-Yves; Jessen, Katti A.; Tang, Crystal M.; Huser, Nanni H.; Felce, Jeremy D.; Froning, Karen J.; Peterman, Marshall C.; Aubol, Brandon E.; Gessert, Steve F.; Sauder, J. Michael; Schwinn, Kenneth D.; Russell, Marijane; Rooney, Isabelle A.; Adams, Jason; Leon, Barbara C.; Do, Tuan H.; Blaney, Jeff M.; Sprengeler, Paul A.; Thompson, Devon A.; Smyth, Lydia; Pelletier, Laura A.; Atwell, Shane; Holme, Kevin; Wasserman, Stephen R.; Emtage, Spencer; Burley, Stephen K.; Reich, Siegfried H.; (Ventana); (SGX); (Genentech); (Amira)

    2010-01-12

    The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC{sub 50} of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition.

  2. Spleen tyrosine kinase mediates high glucose-induced transforming growth factor-{beta}1 up-regulation in proximal tubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Won Seok; Chang, Jai Won [Division of Nephrology, Department of Internal Medicine, Asan Medical Center, College of Medicine, University of Ulsan, Seoul (Korea, Republic of); Han, Nam Jeong [Department of Cell Biology, Asan Institute for Life Sciences, Seoul (Korea, Republic of); Lee, Sang Koo [Division of Nephrology, Department of Internal Medicine, Asan Medical Center, College of Medicine, University of Ulsan, Seoul (Korea, Republic of); Park, Su-Kil, E-mail: skpark@amc.seoul.kr [Division of Nephrology, Department of Internal Medicine, Asan Medical Center, College of Medicine, University of Ulsan, Seoul (Korea, Republic of)

    2012-09-10

    The role of spleen tyrosine kinase (Syk) in high glucose-induced intracellular signal transduction has yet to be elucidated. We investigated whether Syk is implicated in high glucose-induced transforming growth factor-{beta}1 (TGF-{beta}1) up-regulation in cultured human proximal tubular epithelial cells (HK-2 cell). High glucose increased TGF-{beta}1 gene expression through Syk, extracellular signal-regulated kinase (ERK), AP-1 and NF-{kappa}B. High glucose-induced AP-1 DNA binding activity was decreased by Syk inhibitors and U0126 (an ERK inhibitor). Syk inhibitors suppressed high glucose-induced ERK activation, whereas U0126 had no effect on Syk activation. High glucose-induced NF-{kappa}B DNA binding activity was also decreased by Syk inhibitors. High glucose increased nuclear translocation of p65 without serine phosphorylation of I{kappa}B{alpha} and without degradation of I{kappa}B{alpha}, but with an increase in tyrosine phosphorylation of I{kappa}B{alpha} that may account for the activation of NF-{kappa}B. Both Syk inhibitors and Syk-siRNA attenuated high glucose-induced I{kappa}B{alpha} tyrosine phosphorylation and p65 nuclear translocation. Depletion of p21-activated kinase 2 (Pak2) by transfection of Pak2-siRNA abolished high glucose-induced Syk activation. In summary, high glucose-induced TGF-{beta}1 gene transcription occurred through Pak2, Syk and subsequent ERK/AP-1 and NF-{kappa}B pathways. This suggests that Syk might be implicated in the diabetic kidney disease.

  3. Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Carrasco-Garcia, Estefania; Saceda, Miguel [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche (Alicante) (Spain); Grasso, Silvina; Rocamora-Reverte, Lourdes; Conde, Mariano; Gomez-Martinez, Angeles [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Garcia-Morales, Pilar [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche (Alicante) (Spain); Ferragut, Jose A. [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Martinez-Lacaci, Isabel, E-mail: imlacaci@umh.es [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad AECC de Investigacion Traslacional en Cancer, Hospital Universitario Virgen de la Arrixaca, 30120 Murcia (Spain)

    2011-06-10

    Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G{sub 1} arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G{sub 1} arrest. This G{sub 1} arrest was associated with up-regulation of p27{sup kip1}, whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G{sub 1} arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 {Delta}EGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.

  4. Preclinical evaluation of sunitinib, a multi-tyrosine kinase inhibitor, as a radiosensitizer for human prostate cancer

    Directory of Open Access Journals (Sweden)

    Brooks Colin

    2012-09-01

    Full Text Available Abstract Background Many prostate cancers demonstrate an increased expression of growth factor receptors such as vascular endothelial growth factor receptor (VEGFR and platelet derived growth factor receptor (PDGFR which have been correlated with increased resistance to radiotherapy and poor prognosis in other tumors. Therefore, response to radiation could potentially be improved by using inhibitors of these abnormally activated pathways. We have investigated the radiosensitizing effects of sunitinib, a potent, multi-tyrosine kinase inhibitor of the VEGFR and PDGFR receptors, on human prostate cancer cells. Methods The radiosensitizing effects of sunitinib were assessed on human prostate cancer cell lines DU145, PC3 and LNCaP by clonogenic assay. Sunitinib’s ability to inhibit the activities of its key targets was determined by immunoblot analysis. The radiosensitizing effects of sunitinib in vivo were tested on human tumor xenografts growing in nude mice where response was assessed by tumor growth delay. Results Clonogenic survival curve assays for both DU145 and PC3 cells showed that the surviving fraction at 2 Gy was reduced from 0.70 and 0.52 in controls to 0.44 and 0.38, respectively, by a 24 hr pretreatment with 100 nM sunitinib. LNCaP cells were not radiosensitized by sunitinib. Dose dependent decreases in VEGFR and PDGFR activation were also observed following sunitinib in both DU145 and PC3 cells. We assessed the ability of sunitinib to radiosensitize PC3 xenograft tumors growing in the hind limb of nude mice. Sunitinib given concurrently with radiation did not prolong tumor growth delay. However, when animals were treated with sunitinib commencing the day after fractionated radiation was complete, tumor growth delay was enhanced compared to radiation alone. Conclusions We conclude, based on the in vivo results, that sunitinib and radiation do not interact directly to radiosensitize the PC3 tumor cells in vivo as they did in vitro

  5. A new solvate of afatinib, a specific inhibitor of the ErbB family of tyrosine kinases

    Science.gov (United States)

    Zeller, Matthias; de Araujo, Gabriel Lima Barros; Parker, Trev; Singh Rai, Amrinder; Byrn, Stephen R.

    2017-01-01

    Afatinib (systematic name: N-{4-(3-chloro-4-fluoro­anilino)-7-[(tetra­hydro­furan-3-yl)­oxy]quinazolin-6-yl}-4-(di­methyl­amino)­but-2-enamide), is a specific in­hibitor of the ErbB family of tyrosine kinases. The free base form crystallizes from aceto­nitrile as a mixed water–aceto­nitrile solvent, C24H25ClFN5O3·0.25C2H3N·2H2O. It crystallizes with two independent mol­ecules (A and B) in the asymmetric unit of the chiral space group P4212, but exhibits close to perfect pseudo-inversion symmetry, emulating P4/ncc that relates the two mol­ecules to each other. Exact inversion symmetry is however broken by swapping of oxygen and CH2 moieties of the outer tetra­hydro­furanyl substituents of the two independent mol­ecules. This can, in turn, be traced back to C—H⋯N and C—H⋯O inter­actions of the aceto­nitrile solvent mol­ecules with the tetra­hydro­furan oxygen and CH2 units. In the crystal, neighboring mol­ecules are connected via N—H⋯O hydrogen bonds between the secondary amine and the amide keto O atom. Additional hydrogen bonds are formed through the water solvent mol­ecules, which are engaged in O—H⋯O and O—H⋯N hydrogen bonds connecting to the di­methyl­amino N atom, the amide keto O atom, and one of the quinazoline N atoms of a neighboring mol­ecule, leading to an intricate three-dimensional hydrogen-bonded superstructure. There are two types of channels stretching along the direction of the c axis; one along the fourfold rotational axis, occupied by aceto­nitrile solvent mol­ecules situated on that axis, and parallel channels which are not occupied by any solvent.

  6. Effects of tyrosine kinase inhibitors on spermatogenesis and pituitary gonadal axis in males with chronic myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Yassin MA

    2014-08-01

    Full Text Available Objective: The introduction of several classes of targeted therapeutics for the treatment of chronic myelogenous leukemia (CML raises the question of whether male fertility is affected and the degree of this affection, if any, among the different generations of tyrosine kinase inhibitors (TKIs. Additionally, when two drugs are equally effective, the drug with less toxic effect on fertility is favourable. Our aims were to evaluate semen parameters and pituitary gonadal function before and four months after starting TKIs namely, dasatinib, nilotinib, and imatinib in patients with CML. Design: Prospective study. Setting, patients and interventions: We studied the effect of TKIs' first generation (imatinib and second generation (dasatinib and nilotinib on semen parameters and endocrine functions in 20 eugonadal male patients with CML, aged between 35 to 51 years. They were receiving imatinib (400 mg once daily, dasatinib (100 mg once daily or nilotinib (300 mg twice daily as upfront therapy. We assessed the serum gonadotropins (LH and FSH and testosterone (T secretion and sperm parameters before and after four months of using these TKIs. Results: Four months after starting TKIs, serum testosterone, LH and FSH concentrations decreased significantly. The total sperm count (SC, total and rapid progressive sperm motility, and % sperms with normal morphology decreased significantly versus pre-treatment. After 4 months of therapy, dasatinib had comparatively the least deleterious effects on SC, ejaculate volume (SV, sperm motility and % of sperms with normal morphology (%NM compared to imatinib and nilotinib. Significant correlations were found between serum T concentrations and semen parameters before and after TKIs therapy including SC (r = 0.658 and r = 0.73 respectively, p < 0.001, rapid progressive motility (r = 0.675 and r = 0.758, respectively; p < 0.001, and the % NM (r = 0.752 and r = 0.834, respectively; p < 0.001. After TKIs therapy, LH were

  7. Molecular predictors of response to tyrosine kinase inhibitors in patients with Non-Small-Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Murray Samuel

    2012-09-01

    Full Text Available Abstract Introduction Epidermal growth factor receptor (EGFR tyrosine kinase inhibitors (TKIs have become a treatment option in non-small-cell lung cancer (NSCLC patients. However, despite their use in this disease, a significant number of patients will eventually develop resistance and relapse. In this study, we aimed to characterize several molecular events involved in potential resistance mechanisms to anti-EGFR treatment and correlate our findings with clinical outcome. Material and methods The medical records of patients with NSCLC who received anti-EGFR TKIs in any line within the participating centers were reviewed and available paraffin embedded tissue was retrieved. Mutational analysis for EGFR, KRAS, BRAF and intron-exon 14 deletions of MET; FISH analysis for chromosomal gain or amplification for EGFR, MET and the deletion marker D7S486 were performed. Furthermore, the expression of EGFR and MET were analysed by immunohistochemistry. All results were correlated with treatment outcomes. Results Between 10/2001 and 12/2009 from an initial cohort of 72 treated patients, 59 cases (28 gefitinib/ 31 erlotinib were included in the analysis. The majority had adenocarcinoma histology (68%, and received treatment in the second line setting (56%. Disease control rate (DCR was 25.4% for all patients. EGFR and RAS mutational rates were 33% and 10% respectively, no other mutations were identified. High EGFR expressing tumors were found in 7 of 45 cases and pEGFR positivity (IHC was found in 56% of the cases; MET expression was found in 48% of tumors. EGFR gene amplification was found in 4 cases, two cases showed high polysomy; overall, 13% cases were FISH positive for EGFR. High polysomy of MET gene was detected in 1/43 cases tested. D7S486 locus deletion was detected in 15/37 (40% of cases. EGFR mutational status and gene gain were both associated with more favorable DCR. No other associations between examined biomarkers and DCR or survival were

  8. Radiation Response Modulation of GW572016 (EGFR/HER2 Dual Tyrosine Kinase Inhibitor) in Human Breast Cancer Xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yeon Sil; Roh, Kwang Won; Chae, Soo Min; Yoon, Sei Chul; Jang, Hong Seok; Chung, Su Mi [The Catholic University of Korea, College of Medicine, Seoul (Korea, Republic of); Mun, Seong Kwon [Eulji University Hospital, Daejeon (Korea, Republic of)

    2007-12-15

    Purpose: We examined the effect of the dual EGFR/HER2 tyrosine kinase inhibitor, GW572016, on EGFR/HER2 receptor phosphorylation, inhibition of downstream signaling and radiosensitization in either an EGFR or HER2 overexpressing human breast cancer xenograft. Materials and Methods: We established SCID mice xenografts from 4 human breast cancer cell line that overexpressed EGFR or HER 2 (SUM 102, SUM 149, SUM 185, SUM 225). Two series of xenografts were established. One series was established for determining inhibition of the EGFR/HER2 receptor and downstream signaling activities by GW572016. The other series was established for determining the radiosensitization effect of GW572016. Inhibition of the receptor and downstream signaling proteins were measured by the use of immunoprecipitation and Western blotting. For determining the in vivo radiosensitization effect of GW572016, we compared tumor growth delay curves in the following four treatment arms: a) control; b) GW572016 alone; c) radiotherapy (RT) alone; d) GW572016 and RT. Results: GW572016 inhibited EGFR, HER2 receptor phosphorylation in SUM 149 and SUM 185 xenografts. In addition, the p44/42 MAPK (ERK 1/2) downstream signaling pathway was inactivated by GW572016 in the SUM 185 xenograft. In the SUM 225 xenograft, we could not observe inhibition of HER2 receptor phosphorylation by GW572016; both p44/42 MAPK (Erk1/2) and Akt downstream signal protein phosphorylation were inhibited by GW572016. GW572016 inhibited growth of the tumor xenograft of SUM 149 and SUM 185. The combination of GW572016 and RT enhanced growth inhibition greater than that with GW572016 alone or with RT alone in the SUM 149 xenograft. GW572016 appears to act as an in vivo radiosensitizer. Conclusion: GW572016 inhibited EGFR/HER2 receptor phosphorylation and downstream signaling pathway proteins. GW572016 modestly inhibited the growth of tumor in the SUM 185 xenograft and showed radiosensitization in the SUM 149 xenograft. Our results

  9. Regulation of alternative macrophage activation in the liver following acetaminophen intoxication by stem cell-derived tyrosine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, Carol R., E-mail: cgardner@pharmacy.rutgers.edu [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States); Hankey, Pamela [Department of Veterinary and Biomedical Science, Pennsylvania State University, University Park, PA 16802 (United States); Mishin, Vladimir; Francis, Mary [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States); Yu, Shan [Department of Veterinary and Biomedical Science, Pennsylvania State University, University Park, PA 16802 (United States); Laskin, Jeffrey D. [Department of Environmental and Occupational Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854 (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States)

    2012-07-15

    Stem cell-derived tyrosine kinase (STK) is a transmembrane receptor reported to play a role in macrophage switching from a classically activated/proinflammatory phenotype to an alternatively activated/wound repair phenotype. In the present studies, STK{sup −/−} mice were used to assess the role of STK in acetaminophen-induced hepatotoxicity as evidence suggests that the pathogenic process involves both of these macrophage subpopulations. In wild type mice, centrilobular hepatic necrosis and increases in serum transaminase levels were observed within 6 h of acetaminophen administration (300 mg/kg, i.p.). Loss of STK resulted in a significant increase in sensitivity of mice to the hepatotoxic effects of acetaminophen and increased mortality, effects independent of its metabolism. This was associated with reduced levels of hepatic glutathione, rapid upregulation of inducible nitric oxide synthase, and prolonged induction of heme oxygenase-1, suggesting excessive oxidative stress in STK{sup −/−} mice. F4/80, a marker of mature macrophages, was highly expressed on subpopulations of Kupffer cells in livers of wild type, but not STK{sup −/−} mice. Whereas F4/80{sup +} macrophages rapidly declined in the livers of wild type mice following acetaminophen intoxication, they increased in STK{sup −/−} mice. In wild type mice hepatic expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-12, products of classically activated macrophages, increased after acetaminophen administration. Monocyte chemotactic protein-1 (MCP-1) and its receptor, CCR2, as well as IL-10, mediators involved in recruiting and activating anti-inflammatory/wound repair macrophages, also increased in wild type mice after acetaminophen. Loss of STK blunted the effects of acetaminophen on expression of TNFα, IL-1β, IL-12, MCP-1 and CCR2, while expression of IL-10 increased. Hepatic expression of CX3CL1, and its receptor, CX3CR1 also increased in STK{sup −/−} mice

  10. Sequential treatment of tyrosine kinase inhibitors and chemotherapy for EGFR-mutated non-small cell lung cancer: a meta-analysis of Phase III trials

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2013-11-01

    Full Text Available Yiliang Zhang,1,* Yihua Sun,1,* Lei Wang,1 Ting Ye,1 Yunjian Pan,1 Haichuan Hu,1 Yongfu Yu,2 Naiqing Zhao,2 Yanyan Song,3 David Garfield,4 Haiquan Chen1 1Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, 2Department of Biostatistics, School of Public Health, Fudan University, 3Department of Pharmacology and Biostatistics, Institute of Medical Science, Shanghai Jiaotong University School of Medicine, 4ProMed Cancer Centers, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: This aim of this study was to compare the efficacy of first-line tyrosine kinase inhibitor therapy followed, upon progression, by chemotherapy with the reverse sequence in patients with EGFR-mutated non-small cell lung cancer (NSCLC in terms of overall survival. Methods: We performed a meta-analysis of studies that met the following criteria: Phase III clinical trial comparing the sequencing of epidermal growth factor receptor (EGFR tyrosine kinase inhibitors with chemotherapy in the treatment of advanced EGFR-mutated NSCLC; activating mutations reported; and availability of hazard ratio estimates with 95% confidence intervals (CIs for overall survival. Results: Six clinical trials were included in this study. The pooled hazard ratio for overall survival of the EGFR-mutated population that completed sequential treatment was 1.03 (95% CI 0.86–1.22, P=0.776. There was no statistically significant heterogeneity between the studies (tau2 =0; I2=0, 95% CI 0–0.37, P=0.548. Evidence of marked publication bias for the two treatment sequences was insufficient (P=0.145. Conclusion: In patients with advanced NSCLC and activating EGFR mutations, first-line chemotherapy followed upon progression by a tyrosine kinase inhibitor was not inferior in terms of overall survival compared with the inverse sequence. This may serve as an indication that

  11. Research Progress on Resistance Mechanisms of Epidermal Growth Factor Receptor 
Tyrosine Kinase Inhibitors in Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Yuan LI

    2012-02-01

    Full Text Available With a greater understanding of tumor biology, novel molecular-targeted strategies that block cancer progression pathways have been evaluated as a new therapeutic approach for treating non-small cell lung cancer (NSCLC. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs, such as gefitinib and erlotinib, show favorable response to EGFR mutant lung cancer in some populations of NSCLC patients. However, the efficacy of EGFR-TKIs is limited by either primary (de novo or acquired resistance after therapy. This review will focus on recently identified mechanisms of primary and acquired resistance to EGFR TKIs and strategies currently being employed to overcome resistance.

  12. Spleen Tyrosine Kinase Inhibitor Fostamatinib Disodium for the Treatment of Rheumatoid Arthritis%类风湿性关节炎治疗药Fostamatinib disodium

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    @@ 脾酪氨酸激酶(spleen tyrosine kinase,SYK) 为一种非受体型酪氨酸蛋白激酶,具有广泛的生物活性,其在免疫细胞的成熟和激活中发挥着重要作用,现已被证明与类风湿性关节炎(rheumatoid arthritis,RA) 的发生、发展密切相关.

  13. Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins.

    OpenAIRE

    1993-01-01

    Cross-linking of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins on T cells can trigger cell activation. We and others have shown an association between GPI-anchored proteins and the protein tyrosine kinases (PTKs) p56lck and p59fyn, suggesting a pathway for signaling through GPI-anchored proteins. Studies of decay-accelerating factor (DAF) or CD59 in either the C32 cell line or the HeLa cell line transfected with PTK cDNA demonstrated that the GPI-anchored proteins associated ...

  14. Etk/Bmx, a PH-domain containing tyrosine kinase, protects prostate cancer cells from apoptosis induced by photodynamic therapy or thapsigargin.

    Science.gov (United States)

    Xue, L Y; Qiu, Y; He, J; Kung, H J; Oleinick, N L

    1999-06-03

    Prostate carcinoma (PCA) is the most frequently diagnosed malignancy in American men. PCA at advanced stages can both proliferate abnormally and resist apoptosis. Among the many known signal transduction pathways, phosphatidylinositide-3'OH kinase (PI3-kinase) has been shown to play an important role in cell survival and resistance to apoptosis. In this study, we investigate the involvement of Etk/Bmx, a newly discovered tyrosine kinase that is a substrate of PI3-kinase, in protection of prostate cancer cells from apoptosis. Parental LNCaP cells and two derivative cell lines, one overexpressing wild type Etk (Etkwt) and the other expressing a dominant negative Etk (EtkDN), were used to study the function of Etk. The cells were treated with photodynamic therapy (PDT), a newly approved cancer treatment which employs a photosensitizer and visible light to produce an oxidative stress in cells, often leading to apoptosis. Our results indicate that PDT induces apoptosis in LNCaP cells, as measured by DNA fragmentation and by cleavage of poly(ADP-ribose) polymerase (PARP), and moreover, the extent of apoptosis was much reduced in Etkwt cells as compared to LNCaP or EtkDN cells. Assay of overall cell viability confirmed that Etkwt cells were considerably less sensitive to PDT than were the parental LNCaP or EtkDN cells. Similar results were found in response to thapsigargin (TG). A specific inhibitor of PI3-kinase, LY294002, abolished Etk activity and markedly increased TG-induced PARP cleavage. The results suggest that Etk/Bmx is an efficient effector of PI3-kinase and that the newly described PI3-kinase/Etk pathway is involved in the protection of prostate carcinoma cells from apoptosis in response to PDT or TG.

  15. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Energy Technology Data Exchange (ETDEWEB)

    Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  16. Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H{sub 2}O{sub 2} in HL-1 mouse cardiac muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Rao, F. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Deng, C.Y. [Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Zhang, Q.H.; Xue, Y.M. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Xiao, D.Z.; Kuang, S.J.; Lin, Q.X.; Shan, Z.X.; Liu, X.Y.; Zhu, J.N. [Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Yu, X.Y. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Wu, S.L. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China)

    2013-09-06

    Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H{sub 2}O{sub 2}), but not angiotensin II, stimulated MIF expression in HL-1 cells. H{sub 2}O{sub 2}-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H{sub 2}O{sub 2}-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

  17. EGFR tyrosine kinase targeted compounds: in vitro antitumor activity and molecular modeling studies of new benzothiazole and pyrimido[2,1-b]benzothiazole derivatives.

    Science.gov (United States)

    Gabr, Moustafa T; El-Gohary, Nadia S; El-Bendary, Eman R; El-Kerdawy, Mohamed M

    2014-01-01

    In this study, we illustrate computer aided drug design of new benzothiazole and pyrimido[2,1-b]benzothiazole derivatives as epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors. Compounds 1-5 were screened at NCI, USA, for antitumor activity against non-small cell lung cancer (NCI-H522), colon cancer (HCT-116, HCT-15 and HT29) and breast cancer (MDA-MB-468 and MDA-MB-231/ATCC) cell lines in which EGFR is overexpressed in varying levels. Results indicated that these compounds are more potent antitumor agents compared to erlotinib against HT29 and MDA-MB-231/ATCC cell lines. Compound 3 showed GI50 value of 22.3 nM against NCI-H522 cell line, while erlotinib exhibited GI50 value of 1 µM against the same cell line. In addition, these compounds were studied for their EGFR tyrosine kinase inhibitory activity. Virtual screening utilizing molecular modeling and QSAR techniques enabled the understanding of the pharmacophoric requirements for antitumor activity. Docking the designed compounds into the ATP binding site of EGFR-TK domain was done to predict the analogous binding mode of these compounds to the EGFR-TK inhibitors.

  18. The role of tyrosine kinase Etk/Bmx in EGF-induced apoptosis of MDA-MB-468 breast cancer cells.

    Science.gov (United States)

    Chen, Kai-Yun; Huang, Li-Ming; Kung, Hsing-Jien; Ann, David K; Shih, Hsiu-Ming

    2004-03-11

    Etk/Bmx, a member of the Tec family of tyrosine kinases, mediates various signaling pathways and confers several cellular functions. In the present study, we have explored the functional role of Etk in mediating EGF-induced apoptosis, using MDA-MB-468 cell line as a model. We first demonstrated that EGF treatment induces Etk tyrosine phosphorylation in both HeLa and MDA-MB-468 cells. Overexpression of Etk by recombinant adenovirus in MDA-MB-468 cells potentiates the extent of EGF-induced cell apoptosis. The observed Etk-enhanced MDA-MB-468 cell apoptosis is associated with the Stat1 activation, as demonstrated by electrophoresis mobility shift assays and reporter gene assays. By contrast, a kinase domain deletion mutant EtkDeltaK, functioning as a dominant-negative mutant, ameliorates EGF-induced Stat1 activation and apoptosis in MDA-MB-468 cells. To explore whether the activated Etk alone is sufficient for inducing apoptosis, a conditionally activated Etk (DeltaEtk-ER), a chimeric fusion protein of PH domain-truncated Etk and ligand-binding domain of estrogen receptor, was introduced into MDA-MB-468 cells. Upon beta-estradiol ligand activation, the DeltaEtk-ER could stimulate Stat1 activity and confer cell apoptosis independent of EGF treatment. Taken together, our findings indicate that Etk is a downstream signaling molecule of EGF receptor and suggest that Etk activation is essential for transducing the EGF-induced apoptotic signaling.

  19. Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

    Science.gov (United States)

    Pal Sharma, C; Goldmann, Wolfgang H

    2004-01-01

    Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

  20. The human epidermal growth factor receptor (EGFR gene in European patients with advanced colorectal cancer harbors infrequent mutations in its tyrosine kinase domain

    Directory of Open Access Journals (Sweden)

    Delvenne Philippe

    2011-10-01

    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR, a member of the ErbB family of receptors, is a transmembrane tyrosine kinase (TK activated by the binding of extracellular ligands of the EGF-family and involved in triggering the MAPK signaling pathway, which leads to cell proliferation. Mutations in the EGFR tyrosine kinase domain are frequent in non-small-cell lung cancer (NSCLC. However, to date, only very few, mainly non-European, studies have reported rare EGFR mutations in colorectal cancer (CRC. Methods We screened 236 clinical tumor samples from European patients with advanced CRC by direct DNA sequencing to detect potential, as yet unknown mutations, in the EGFR gene exons 18 to 21, mainly covering the EGFR TK catalytic domain. Results EGFR sequences showed somatic missense mutations in exons 18 and 20 at a frequency of 2.1% and 0.4% respectively. Somatic SNPs were also found in exons 20 and 21 at a frequency of about 3.1% and 0.4% respectively. Of these mutations, four have not yet been described elsewhere. Conclusions These mutation frequencies are higher than in a similarly sized population characterized by Barber and colleagues, but still too low to account for a major role played by the EGFR gene in CRC.

  1. Overexpression of Tyro3 receptor tyrosine kinase leads to the acquisition of taxol resistance in ovarian cancer cells.

    Science.gov (United States)

    Lee, Chuhee

    2015-07-01

    The majority of patients with ovarian cancer are diagnosed at the advanced stages (III/IV) and their 5-year-survival rate is relatively low. One of the major causes of the poor prognosis of ovarian cancer is the development of resistance to first-line chemotherapy, including platinum and taxol. Therefore, improvements in current understanding of chemoresistance is required for the successful treatment of ovarian cancer. In the present study, taxol-resistant ovarian cancer cells, SKOV3/TR, were established by exposing parental SKOV3 cells to increasing concentrations of taxol. . Briefly, cells were treated with 1.5 nM (for 4 weeks), 3 nM (for 4 weeks), 6 nM (for 5 weeks), 12 nM (for 5 weeks) and 24 nM taxol (for 8 weeks) over 6 months. The SKOV3/TR cells were found to be smaller in size and rounder in shape compared with their parental cells. Cell viability and colony formation assays demonstrated an increase in the population doubling time of the SKOV3/TR cells, indicating a reduction in the proliferative capacity of these cells. Reverse transcription-polymerase chain reaction and western blot analysis revealed that, among the TAM receptor tyrosine kinases (RTKs), the mRNA and protein expression levels of Tyro3 RTK were increased, while those of Axl and Mer RTK were decreased in the SKOV3/TR cells. In addition, restoration of the level of Tyro3 by transfecting Tyro3-specific small interfering RNA into the SKOV3/TR cells reduced the proliferative capacity of the cells, indicating that upregulation of the expression of Tyro3 in SKOV3/TR cells may promote survival in the presence of taxol, which eventually resulted in the acquisition of resistance upon taxol treatment. The present study subsequently found that, in the SKOV3/TR cells, the level of intracellular reactive oxygen species (ROS) was elevated, and antioxidant treatment with N-acetyl cysteine (NAC) exerted more profound antiproliferative effects compared with the parental cells. The western blot analysis

  2. Direct interaction of Syk and Lyn protein tyrosine kinases in rat basophilic leukemia cells activated via type I Fc epsilon receptors.

    Science.gov (United States)

    Amoui, M; Dráberová, L; Tolar, P; Dráber, P

    1997-01-01

    Activation of rat mast cells through the receptor with high affinity for IgE (Fc epsilonRI) requires a complex set of interactions involving transmembrane subunits of the Fc epsilonRI and two classes of nonreceptor protein tyrosine kinase (PTK). the Src family PTK p53/p56(lyn) (Lyn) and the Syk/ZAP-family PTK p72(syk) (Syk). Early activation events involve increased activity of Lyn and Syk kinases and their translocation into membrane domains containing aggregated Fc epsilonRI, but the molecular mechanisms responsible for these changes have remained largely unclear. To determine the role of Fc epsilonRI subunits in this process, we have analyzed Syk- and Lyn-associated proteins in activated rat basophilic leukemia (RBL) cells and their variants deficient in the expression of Fc epsilonRI beta or gamma subunits. Sepharose 4B gel chromatography of postnuclear supernatants from Nonidet-P40-solubilized antigen (Ag)- or pervanadate-activated RBL cells revealed extensive changes in the size of complexes formed by Lyn and Syk kinases and other cellular components. A fusion protein containing Src homology 2 (SH2) and SH3 domains of Lyn bound Syk from lysates of nonactivated RBL cells; an increased binding was observed when lysates from Ag- or pervanadate-activated cells were used. A similar amount of Syk was bound when lysates from pervanadate-activated variant cells deficient in the expression of Fc epsilonRI beta or gamma subunits were used, suggesting that Fc epsilonRI does not function as the only intermediate in the formation of the Syk-Lyn complexes. Further experiments have indicated that Syk-Lyn interactions occur in Ag-activated RBL cells under in vivo conditions and that these interactions could involve direct binding of the Lyn SH2 domain with phosphorylated tyrosine of Syk. The physical association of Lyn and Syk during mast-like cell activation supports the recently proposed functional cooperation of these two tyrosine kinases in Fc epsilonRI signaling.

  3. BI 885578, a Novel IGF1R/INSR Tyrosine Kinase Inhibitor with Pharmacokinetic Properties That Dissociate Antitumor Efficacy and Perturbation of Glucose Homeostasis.

    Science.gov (United States)

    Sanderson, Michael P; Apgar, Joshua; Garin-Chesa, Pilar; Hofmann, Marco H; Kessler, Dirk; Quant, Jens; Savchenko, Alexander; Schaaf, Otmar; Treu, Matthias; Tye, Heather; Zahn, Stephan K; Zoephel, Andreas; Haaksma, Eric; Adolf, Günther R; Kraut, Norbert

    2015-12-01

    Inhibition of the IGF1R, INSRA, and INSRB receptor tyrosine kinases represents an attractive approach of pharmacologic intervention in cancer, owing to the roles of the IGF1R and INSRA in promoting cell proliferation and survival. However, the central role of the INSRB isoform in glucose homeostasis suggests that prolonged inhibition of this kinase could result in metabolic toxicity. We describe here the profile of the novel compound BI 885578, a potent and selective ATP-competitive IGF1R/INSR tyrosine kinase inhibitor distinguished by rapid intestinal absorption and a short in vivo half-life as a result of rapid metabolic clearance. BI 885578, administered daily per os, displayed an acce