The Experts below are selected from a list of 339 Experts worldwide ranked by ideXlab platform
Robert Roskoski - One of the best experts on this subject based on the ideXlab platform.
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role of ret protein tyrosine Kinase inhibitors in the treatment ret driven thyroid and lung cancers
Pharmacological Research, 2018Co-Authors: Robert Roskoski, Abdollah SadeghinejadAbstract:Abstract RET is a transmembrane Receptor Protein-Tyrosine Kinase that is required for the development of the nervous system and several other tissues. The mechanism of activation of RET by its glial-cell derived neurotrophic factor (GDNF) ligands differs from that of all other Receptor Protein-Tyrosine Kinases owing to the requirement for additional GDNF family Receptor-α (GFRα) co-Receptors (GFRα1/2/3/4). RET point mutations have been reported in multiple endocrine neoplasia (MEN2A, MEN2B) and medullary thyroid carcinoma. In contrast, RET fusion proteins have been reported in papillary thyroid and non-small cell lung adenocarcinomas. More than a dozen fusion partners of RET have been described in papillary thyroid carcinomas, most frequently CCDC6-RET and NCOA4-RET. RET-fusion proteins, commonly KIF5B-RET, have also been found in non-small cell lung cancer (NSCLC). Several drugs targeting RET have been approved by the FDA for the treatment of cancer: (i) cabozantinib and vandetanib for medullary thyroid carcinomas and (ii) lenvatinib and sorafenib for differentiated thyroid cancers. In addition, alectinib and sunitinib are approved for the treatment of other neoplasms. Each of these drugs is a multiKinase inhibitor that has activity against RET. Previous X-ray studies indicated that vandetanib binds within the ATP-binding pocket and forms a hydrogen bond with A807 within the RET hinge and it makes hydrophobic contact with L881 of the catalytic spine which occurs in the floor of the adenine-binding pocket. Our molecular modeling studies indicate that the other antagonists bind in a similar fashion. All of these antagonists bind to the active conformation of RET and are therefore classified as type I inhibitors. The drugs also make variable contacts with other residues of the regulatory and catalytic spines. None of these drugs was designed to bind preferentially to RET and it is hypothesized that RET-specific antagonists might produce even better clinical outcomes. Currently the number of new cases of neoplasms bearing RET mutations or RET-fusion proteins is estimated to be about 10,000 per year in the United States. This is about the same as the incidence of chronic myelogenous leukemia for which imatinib and second and third generation BCR-Abl non-Receptor Protein-Tyrosine Kinase antagonists have proven clinically efficacious and which are commercially successful. These findings warrant the continued development of specific antagonists targeting RET-driven neoplasms.
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anaplastic lymphoma Kinase alk inhibitors in the treatment of alk driven lung cancers
Pharmacological Research, 2017Co-Authors: Robert RoskoskiAbstract:Anaplastic lymphoma Kinase is expressed in two-thirds of the anaplastic large-cell lymphomas as an NPM-ALK fusion protein. Physiological ALK is a Receptor Protein-Tyrosine Kinase within the insulin Receptor superfamily of proteins that participates in nervous system development. The EML4-ALK fusion protein and four other ALK-fusion proteins play a fundamental role in the development in about 5% of non-small cell lung cancers. The amino-terminal portions of the ALK fusion proteins result in dimerization and subsequent activation of the ALK protein Kinase domain that plays a key role in the pathogenesis of various tumors. Downstream signaling from the ALK fusion protein leads to the activation of the Ras/Raf/MEK/ERK1/2 cell proliferation module and the JAK/STAT cell survival pathways. Moreover, nearly two dozen ALK activating mutations are involved in the pathogenesis of childhood neuroblastomas. The occurrence of oncogenic ALK-fusion proteins, particularly in non-small cell lung cancer, has fostered considerable interest in the development of ALK inhibitors. Crizotinib was the first such inhibitor approved by the US Food and Drug Administration for the treatment of ALK-positive non-small cell lung cancer in 2011. The median time for the emergence of crizotinib drug resistance is 10.5 months after the initiation of therapy. Such resistance prompted the development of second-generation drugs including ceritinib and alectinib, which are approved for the treatment of non-small cell lung cancer. Unlike the single gatekeeper mutation that occurs in drug-resistant epidermal growth factor Receptor in lung cancer, nearly a dozen different mutations in the catalytic domain of ALK fusion proteins have been discovered that result in crizotinib resistance. Crizotinib, ceritinib, and alectinib form a complex within the front cleft between the small and large lobes of an inactive ALK protein-Kinase domain with a compact activation segment. These drugs are classified as type I½ B inhibitors because they bind to an inactive enzyme and they do not extend past the gatekeeper into the back pocket of the drug binding site.
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anaplastic lymphoma Kinase alk structure oncogenic activation and pharmacological inhibition
Pharmacological Research, 2013Co-Authors: Robert RoskoskiAbstract:Anaplastic lymphoma Kinase was first described in 1994 as the NPM-ALK fusion protein that is expressed in the majority of anaplastic large-cell lymphomas. ALK is a Receptor Protein-Tyrosine Kinase that was more fully characterized in 1997. Physiological ALK participates in embryonic nervous system development, but its expression decreases after birth. ALK is a member of the insulin Receptor superfamily and is most closely related to leukocyte tyrosine Kinase (Ltk), which is a Receptor Protein-Tyrosine Kinase. Twenty different ALK-fusion proteins have been described that result from various chromosomal rearrangements, and they have been implicated in the pathogenesis of several diseases including anaplastic large-cell lymphoma, diffuse large B-cell lymphoma, and inflammatory myofibroblastic tumors. The EML4-ALK fusion protein and four other ALK-fusion proteins play a fundamental role in the development in about 5% of non-small cell lung cancers. The formation of dimers by the amino-terminal portion of the ALK fusion proteins results in the activation of the ALK protein Kinase domain that plays a key role in the tumorigenic process. Downstream signaling from ALK fusion proteins involves the Ras/Raf/MEK/ERK1/2 cell proliferation module and the JAK/STAT cell survival pathway. Furthermore, nearly two dozen ALK activating mutations participate in the pathogenesis of childhood neuroblastomas along with ALK overexpression. The occurrence of oncogenic ALK, particularly in non-small cell lung cancer, has generated considerable interest and effort in developing ALK inhibitors. Currently, crizotinib has been approved by the US Food and Drug Administration for the treatment of ALK-positive non-small cell lung cancer along with an approved fluorescence in situ hybridization kit used for the diagnosis of the disease. The emergence of crizotinib drug resistance with a median occurrence at approximately 10 months after the initiation of therapy has stimulated the development of second-generation drugs for the treatment of non-small cell lung cancer and other disorders. About 28% of the cases of crizotinib resistance are related to nearly a dozen different mutations of ALK in the EML4-ALK fusion protein; the other cases of resistance are related to the upregulation of alternative signaling pathways or to undefined mechanisms. It is remarkable that the EML4-ALK fusion protein was discovered in 2007 and crizotinib was approved for the treatment of ALK-positive non-small cell lung cancer in 2011, which is a remarkably short timeframe in the overall scheme of drug discovery.
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structure and regulation of kit protein tyrosine Kinase the stem cell factor Receptor
Biochemical and Biophysical Research Communications, 2005Co-Authors: Robert RoskoskiAbstract:Abstract Signaling by stem cell factor and Kit, its Receptor, play important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a glycoprotein Receptor Protein-Tyrosine Kinase. The complete absence of stem cell factor or Kit is lethal. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, mastocytomas, and nasal T-cell lymphomas. Binding of stem cell factor to Kit results in Receptor dimerization and activation of protein Kinase activity. The activated Receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. Kit activates Akt, Src family Kinases, phosphatidylinositol 3-Kinase, phospholipase Cγ, and Ras/mitogen-activated protein Kinases. Kit exists in active and inactive conformations as determined by X-ray crystallography. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane domain, and a protein Kinase domain that contains an insert of about 80 amino acid residues. The juxtamembrane domain inhibits enzyme activity in cis by maintaining the control αC-helix and the activation loop in their inactive conformations. The juxtamembrane domain also inhibits Receptor dimerization. STI-571, a clinically effective targeted Protein-Tyrosine Kinase inhibitor, binds to an inactive conformation of Kit. The majority of human gastrointestinal stromal tumors have Kit gain-of-function mutations in the juxtamembrane domain, and most people with these tumors respond to STI-571. STI-571 binds to Kit and Bcr-Abl (the oncoprotein of chronic myelogenous leukemia) at their ATP-binding sites.
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signaling by kit protein tyrosine Kinase the stem cell factor Receptor
Biochemical and Biophysical Research Communications, 2005Co-Authors: Robert RoskoskiAbstract:Signaling by stem cell factor and Kit, its Receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a Receptor Protein-Tyrosine Kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein Kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in Receptor dimerization and activation of protein Kinase activity. The activated Receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family Kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src Kinase homologous Kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the Kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-Kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal Kinase domain binds phosphatidylinositol 3-Kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal Kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of interaction with several enzymes and adaptor proteins.
Won Sik Shin - One of the best experts on this subject based on the ideXlab platform.
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catalytically inactive Receptor tyrosine Kinase ptk7 activates fgfr1 independent of fgf
The FASEB Journal, 2019Co-Authors: Won Sik ShinAbstract:Protein tyrosine Kinase 7 (PTK7), a catalytically defective Receptor protein tyrosine Kinase (RPTK), plays an oncogenic role by activating an unidentified TKI-258 (dovitinib)-sensitive RPTK in esop...
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biphasic regulation of tumorigenesis by ptk7 expression level in esophageal squamous cell carcinoma
Scientific Reports, 2018Co-Authors: Won Sik ShinAbstract:Protein tyrosine Kinase 7 (PTK7), also known as colon carcinoma Kinase 4 (CCK-4), is a member of the catalytically defective Receptor protein tyrosine Kinase family and is upregulated in various cancers, where it is known to act as either an oncoprotein or a tumor suppressor. To understand the contrasting roles of PTK7 in tumorigenesis, we analyzed the tumorigenic characteristics of esophageal squamous cell carcinoma (ESCC) cells with low levels of endogenous PTK7 expression (TE-5 and TE-14 cells) and high levels of expression (TE-6 and TE-10 cells) after transfections with a PTK7 expression vector. PTK7 overexpression increased the proliferation of TE-5 and TE-14 cells but decreased the proliferation of TE-6 and TE-10 cells. In the ESCC cells, proliferation, migration, and invasion were initially increased and then decreased according to PTK7 expression levels, which were mirrored by initial increases and then decreases in the tyrosine phosphorylation of cellular proteins and phosphorylation of Src, Akt, and ERK. In ESCC patients included in The Cancer Genome Atlas database, those with higher PTK7 mRNA levels had a longer overall survival and lower relative risk than those with lower PTK7 mRNA levels. These results demonstrate that PTK7 biphasically regulates tumorigenesis in ESCC.
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catalytically defective Receptor protein tyrosine Kinase ptk7 enhances invasive phenotype by inducing mmp 9 through activation of ap 1 and nf κb in esophageal squamous cell carcinoma cells
Oncotarget, 2016Co-Authors: Won Sik Shin, Yuri HongAbstract:// Won-Sik Shin 1 , Yuri Hong 1 , Hae Won Lee 2 , Seung-Taek Lee 1 1 Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea 2 Department of Thoracic Surgery, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea Correspondence to: Seung-Taek Lee, email: stlee@yonsei.ac.kr Keywords: PTK7, MMP-9, NF-κB, AP-1, esophageal squamous cell carcinoma (ESCC) Received: June 04, 2016 Accepted: September 21, 2016 Published: September 28, 2016 ABSTRACT Protein tyrosine Kinase 7 (PTK7), a member of the catalytically defective Receptor protein tyrosine Kinase family, is upregulated in various cancers including esophageal squamous cell carcinoma (ESCC). Here, we have explored the molecular mechanism of PTK7-dependent invasiveness in ESCC cells. PTK7 knockdown reduced gelatin degradation and MMP-9 secretion in cultures of ESCC TE-10 cells, and showed reduced levels of MMP9 mRNA using real-time RT-PCR and luciferase reporter assays. PTK7 knockdown decreased not only phosphorylation of NF-κB, IκB, ERK, and JNK, but also nuclear localization of NF-κB and AP-1 consisting of c-Fos and c-Jun. Activation of AP-1 and NF-κB requires PTK7-mediated activation of tyrosine Kinases, including Src. In addition, NF-κB activation by PTK7 involves the PI3K/Akt signaling pathway. PTK7-mediated upregulation of MMP9 was also observed in other ESCC cell lines and in three-dimensional cultures of TE-10 cells. Moreover, MMP-9 expression positively correlated with PTK7 expression in ESCC tumor tissue. These findings demonstrate that PTK7 upregulates MMP9 through activation of AP-1 and NF-κB and, thus increases invasive properties of ESCC cells.
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biphasic effect of ptk7 on kdr activity in endothelial cells and angiogenesis
Biochimica et Biophysica Acta, 2015Co-Authors: Won Sik Shin, Hye Won NaAbstract:Abstract Protein tyrosine Kinase 7 (PTK7) is a member of the defective Receptor protein tyrosine Kinase family which lacks catalytic activity. Expression of PTK7 is increased in various cancers but its role in carcinogenesis is not well understood. We previously showed that disruption of PTK7 function suppresses VEGF-induced angiogenic phenotypes in HUVECs and mice. Here, we investigated molecular mechanisms for modulating VEGF-induced physiological effects by PTK7. Treatment with a high concentration of extracellular domain of PTK7 (soluble PTK7; sPTK7) or knockdown of PTK7 inhibited VEGF-induced phosphorylation of Kinase insert domain Receptor (KDR) but did not inhibit phosphorylation of fms-related tyrosine Kinase 1 (FLT-1) in HUVECs. PTK7, more specifically sPTK7, interacted with KDR but not with FLT-1 in HUVECs and HEK293 cells. In vitro binding assay showed that sPTK7 formed oligomers with the extracellular domain of KDR (sKDR) up to an approximately 1:3 molar ratio, and vice versa . sPTK7 at lower molar ratios than sKDR enhanced the binding of VEGF to sKDR. At the same or higher molar ratios, it reduced the binding of VEGF to sKDR. Increasing concentrations of sPTK7 or increasing levels of PTK7 expression first increased and then decreased VEGF-induced KDR phosphorylation, migration, and capillary-like tube formation of HUVECs, as well as in vivo angiogenesis. Taken together, our data demonstrates that PTK7 regulates the activity of KDR biphasically by inducing oligomerization of KDR molecules at lower concentrations and by surrounding KDR molecules at higher concentrations.
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soluble ptk7 inhibits tube formation migration and invasion of endothelial cells and angiogenesis
Biochemical and Biophysical Research Communications, 2008Co-Authors: Won Sik Shin, Yongsun Maeng, Jae-won Jung, Youngguen KwonAbstract:Human PTK7 is a defective Receptor protein tyrosine Kinase and its expression is upregulated in various cancers including colorectal carcinomas. To determine whether PTK7 functions in angiogenesis, we have expressed and purified the extracellular domain of PTK7 (soluble PTK7; sPTK7) as a decoy Receptor to counteract the effects of endogenous PTK7. Capillary-like tube formation of human umbilical vascular endothelial cells (HUVECs) was accompanied by modulation in the PTK7 mRNA level. Neutralization of endogenous PTK7 with sPTK7 inhibited vascular endothelial growth factor (VEGF)-induced tube formation, migration, and invasion of HUVECs in a dose-dependent manner. sPTK7 reduced VEGF-induced phosphorylation of focal adhesion Kinase (FAK) and paxillin, relocalization of paxillin to focal adhesions, and formation of stress fibers. Moreover, sPTK7 inhibited VEGF-induced angiogenesis in vivo. Knockdown of PTK7 using siRNA also inhibited VEGF-induced tube formation, supporting that sPTK7 specifically blocks function of the endogenous PTK7. These results demonstrate that PTK7 plays an important role not only in tube formation, migration, and invasion of endothelial cells but also in angiogenesis.
Pierre Chambon - One of the best experts on this subject based on the ideXlab platform.
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The expression pattern of the mouse Receptor tyrosine Kinase gene MDK1 is conserved through evolution and requires Hoxa-2 for rhombomere-specific expression in mouse embryos.
Developmental biology, 1996Co-Authors: Reshma Taneja, Filippo M Rijli, Bernard Thisse, Christine Thisse, Philippe Bouillet, Pascal Dollé, Pierre ChambonAbstract:Segmentation of the hindbrain has been conserved throughout the vertebrate species and results in the transient formation of rhombomeres, which are lineage-restricted compartments. Studies on the molecular mechanisms underlying the segmentation process have revealed that rhombomeric boundaries coincide with the expression limits of several evolutionary conserved genes such as the zinc-finger transcription factor Krox-20 and homeobox genes which are expressed in a specific spatial and temporal order and have been shown to be important regulators of segmental identity. In addition to Krox-20 and Hox genes, several members of the Eph subfamily of Receptor protein tyrosine Kinase (RTK) genes are also expressed in a segment-restricted manner in the hindbrain, suggesting that these Receptors may act in concert with Hox genes to establish regional identity. In the cascade of regulatory interactions leading to segmental identity, Krox-20 appears to act ‘‘upstream’’ of Hox genes, but the identity of the ‘‘downstream’’ effectors has not yet been identified. We report here the isolation of the zebrafish orthologue of the mouse RTK gene MDK1 which belongs to the Eph Receptor subfamily and show that the major expression domains of the mouse and the zebrafish genes have been conserved through evolution. Since the coincident spatial and temporal expression of Hoxa-2 and MDK1 in the mouse hindbrain suggested a possible regulatory link between them, we analyzed the expression of the MDK1 in Hoxa-2 null mutant embryos. A selective lack of MDK1 expression in rhombomere 3 of Hoxa-2 mutant hindbrains together with an overall altered expression pattern in the other rhombomeres was observed, thus demonstrating that MDK1 lies downstream of Hoxa-2 in the morphogenetic signaling cascade. q 1996 Academic Press, Inc.
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the expression pattern of the mouse Receptor tyrosine Kinase gene mdk1 is conserved through evolution and requires hoxa 2 for rhombomere specific expression in mouse embryos
Developmental Biology, 1996Co-Authors: Reshma Taneja, Filippo M Rijli, Bernard Thisse, Philippe Bouillet, Pascal Dollé, Christine I Thisse, Pierre ChambonAbstract:Abstract Segmentation of the hindbrain has been conserved throughout the vertebrate species and results in the transient formation of rhombomeres, which are lineage-restricted compartments. Studies on the molecular mechanisms underlying the segmentation process have revealed that rhombomeric boundaries coincide with the expression limits of several evolutionary conserved genes such as the zinc-finger transcription factorKrox-20and homeobox genes which are expressed in a specific spatial and temporal order and have been shown to be important regulators of segmental identity. In addition toKrox-20and Hox genes, several members of the Eph subfamily of Receptor protein tyrosine Kinase (RTK) genes are also expressed in a segment-restricted manner in the hindbrain, suggesting that these Receptors may act in concert with Hox genes to establish regional identity. In the cascade of regulatory interactions leading to segmental identity,Krox-20appears to act “upstream” of Hox genes, but the identity of the “downstream” effectors has not yet been identified. We report here the isolation of the zebrafish orthologue of the mouse RTK geneMDK1which belongs to the Eph Receptor subfamily and show that the major expression domains of the mouse and the zebrafish genes have been conserved through evolution. Since the coincident spatial and temporal expression ofHoxa-2andMDK1in the mouse hindbrain suggested a possible regulatory link between them, we analyzed the expression of theMDK1inHoxa-2null mutant embryos. A selective lack ofMDK1expression in rhombomere 3 ofHoxa-2mutant hindbrains together with an overall altered expression pattern in the other rhombomeres was observed, thus demonstrating thatMDK1lies downstream ofHoxa-2in the morphogenetic signaling cascade.
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efficient cloning of cdnas of retinoic acid responsive genes in p19 embryonal carcinoma cells and characterization of a novel mouse gene stra1 mouse lerk 2 eplg2
Developmental Biology, 1995Co-Authors: Philippe Bouillet, Pascal Dollé, Mustapha Ouladabdelghani, Serge Vicaire, Jeanmarie Garnier, Brigitte Schuhbaur, Pierre ChambonAbstract:Abstract Pluripotent mouse P19 embryonal carcinoma (EC) cells have been extensively used as a developmental model system because they can differentiate in the presence of retinoic acid (RA) into derivatives of all three germ layers depending on RA dosage and culture conditions. The expression of several genes has been shown to be induced in RA-treated P19 EC cells and, interestingly, some of these genes may play important roles during mouse embryogenesis. In view of the increasing evidence that RA is a crucial signaling molecule during vertebrate development, we have initiated a study aimed at the systematic isolation of genes whose expression is induced in P19 cells at various times after exposure to RA. We describe here an efficient differential subtractive hybridization cloning strategy which was used to identify additional RA-responsive genes in P19 cells. Fifty different cDNA fragments corresponding to RA-induced genes were isolated. Ten cDNAs represent known genes, 4 of which have already been described as RA-inducible, while the remaining 40 correspond to novel genes. Many of these cDNA sequences represent low-abundance mRNAs. Kinetic analysis of mRNA accumulation following RA treatment allowed us to characterize four classes of RA-responsive genes. We also report the sequence and expression pattern in mouse embryos and adult tissues of one of these novel RA-inducible genes, Stral, and show that it corresponds to the mouse ligand for the Cek5 Receptor Protein-Tyrosine Kinase.
Sandra Pellegrini - One of the best experts on this subject based on the ideXlab platform.
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activation of the protein tyrosine Kinase tyk2 by interferon alpha beta
FEBS Journal, 1994Co-Authors: Giovanna Barbieri, Laura Velazquez, M Fellous, Marina Scrobogna, Sandra PellegriniAbstract:We previously demonstrated that the gene tyk2 rescues the phenotype of a human mutant cell line unresponsive to alpha (IFN) and partially responsive to IFN-beta. Here, we describe functional complementation of the mutant cells with the corresponding cDNA. To characterize the putative non-Receptor protein tyrosine Kinase encoded by the gene tyk2 and begin to understand its functioning, we have raised polyclonal antibodies against a segment of the protein. Using these, we have identified tyk2 as a 134-kDa protein which is rapidly and transiently phosphorylated on tyrosine in response to IFN-alpha/beta and possesses an inducible Kinase activity when tested in vitro. IFN-gamma has no effect on the phosphorylation state of the protein. In agreement with previous genetic evidence, these results assign a role to tyk2 in the IFN-alpha/beta signalling pathway and not in the IFN-gamma pathway. Fractionation of cell lysates have helped to localize the bulk of the protein in the cytoplasm, with a minor fraction associated with the cell membrane. Both protein pools undergo activation upon short-term IFN treatment of intact cells. Through the study of the effect of pervanadate on the phosphorylation level and the activity of tyk2, we conclude that activation of tyk2 by IFN-alpha does not require an intermediate regulatory tyrosine phosphatase.
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a protein tyrosine Kinase in the interferon alpha beta signaling pathway
Cell, 1992Co-Authors: Laura Velazquez, M Fellous, George Stark Stark, Sandra PellegriniAbstract:The mutant human cell line 11.1 is unresponsive to interferon alpha. Here we describe the genetic complementation of this mutant and the identification and cloning of the wild-type gene that corrects the defect. Using transfection with genomic DNA in conjunction with a powerful back-selection, we isolated a cosmid that reverts the mutant phenotype of 11.1 cells. The cosmid encodes a single message whose level is greatly reduced in mutant cells. Complementary DNAs were cloned and found to be virtually identical to tyk2, a human mRNA encoding a non-Receptor protein tyrosine Kinase of previously unknown function. This finding shows that tyk2 links the interferon alpha/beta Receptor to the cytoplasmic transcription factor that mediates activation of interferon-responsive genes.
Gary E Gallick - One of the best experts on this subject based on the ideXlab platform.
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development and characterization of gemcitabine resistant pancreatic tumor cells
Annals of Surgical Oncology, 2007Co-Authors: Ami N Shah, Justin M Summy, Jing Zhang, Serk In Park, Nila U Parikh, Gary E GallickAbstract:BACKGROUND: Pancreatic cancer is an exceptionally lethal disease with an annual mortality nearly equivalent to its annual incidence. This dismal rate of survival is due to several factors including late presentation with locally advanced, unresectable tumors, early metastatic disease, and rapidly arising chemoresistance. To study the mechanisms of chemoresistance in pancreatic cancer we developed two gemcitabine-resistant pancreatic cancer cell lines. METHODS: Resistant cells were obtained by culturing L3.6pl and AsPC-1 cells in serially increasing concentrations of gemcitabine. Stable cultures were obtained that were 40- to 50-fold increased in resistance relative to parental cells. Immunofluorescent staining was performed to examine changes in beta-catenin and E-cadherin localization. Protein expression was determined by immunoblotting. Migration and invasion were determined by modified Boyden chamber assays. Fluorescence-activated cell sorting (FACS) analyses were performed to examine stem cell markers. RESULTS: Gemcitabine-resistant cells underwent distinct morphological changes, including spindle-shaped morphology, appearance of pseudopodia, and reduced adhesion characteristic of transformed fibroblasts. Gemcitabine-resistant cells were more invasive and migratory. Gemcitabine-resistant cells were increased in vimentin and decreased in E-cadherin expression. Immunofluorescence and immunoblotting revealed increased nuclear localization of total beta-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells were activated in the Receptor protein tyrosine Kinase, c-Met and increased in expression of the stem cell markers CD (cluster of differentiation)24, CD44, and epithelial-specific antigen (ESA). CONCLUSIONS: Gemcitabine-resistant pancreatic tumor cells are associated with EMT, a more-aggressive and invasive phenotype in numerous solid tumors. The increased phosphorylation of c-Met may also be related to chemoresistance and EMT and presents as an attractive adjunctive chemotherapeutic target in pancreatic cancer.
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reduced c met expression by an adenovirus expressing a c met ribozyme inhibits tumorigenic growth and lymph node metastases of pc3 ln4 prostate tumor cells in an orthotopic nude mouse model
Clinical Cancer Research, 2003Co-Authors: Sun Jin Kim, Marjorie Johnson, Kristen Koterba, Matthew H Herynk, Hisanori Uehara, Gary E GallickAbstract:Purpose: The expression of c-Met, the Receptor protein tyrosine Kinase for hepatocyte growth factor/scatter factor, frequently increases during prostate tumor progression. However, whether reduced c-Met expression inhibits tumor growth and metastasis has not been ascertained. Experimental Design: c-Met expression was reduced by infection of an adenovirus expressing a c-Met ribozyme into the highly metastatic human prostate cancer cell line PC3-LN4. In vitro , effects on c-Met, Akt, and extracellular signal-regulated Kinase 1/2 expression and phosphorylation, Src expression and activity, and vascular endothelial growth factor expression were determined, as were effects on cell migration and invasion. Prostate tumor formation and metastasis to regional lymph nodes in nude mice were examined after both ex vivo and in vivo infection of cells. Results: Infection of PC3-LN4 cells with the Ad-c-Met-expressing ribozyme decreased steady-state c-Met levels, decreased Src Kinase activity, decreased vascular endothelial growth factor expression, and decreased migration and invasion versus the pU1 (control) virus. Significant inhibition of tumorigenicity (histologically confirmed tumors in only 1 of 10 mice) and consequent lymph node metastasis were observed upon ex vivo infection of Ad-c-Met. Similarly, gene therapy experiments led to complete inhibition of tumor growth in 7 of 8 mice. Conclusions: Reduction in c-Met expression substantially inhibits both tumor growth and lymph node metastasis of PC3-LN4 cells in orthotopic nude mouse models. Therefore, targeting the c-Met signaling pathways may be important in controlling tumor growth and metastasis in human prostate cancers.
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src activation regulates anoikis in human colon tumor cell lines
Oncogene, 2002Co-Authors: Christopher T Windham, Justin M Summy, Nila U Parikh, Doris R Siwak, David J Mcconkey, Alan J Kraker, Gary E GallickAbstract:Src is a non-Receptor protein tyrosine Kinase, the expression and activity of which is increased in >80% of human colon cancers with respect to normal colonic epithelium. Previous studies from this and other laboratories have demonstrated that Src activity contributes to tumorigenicity of established colon adenocarcinoma cell lines. Src participates in the regulation of many signal transduction pathways, among which are those leading to cellular survival. In this study, we addressed the potential role of Src activation to a specific aspect of tumor cell survival, resistance to detachment-induced apoptosis (anoikis). Using five colon tumor cell lines with different biologic properties and genetic alterations, we demonstrate that expression and activity of Src corresponds with resistance to anoikis. Enforced expression of activated Src in subclones of SW480 cells (of low intrinsic Src expression and activity) increases resistance to anoikis; whereas decreased Src expression in HT29 cells (of high Src expression and activity) by transfection with anti-sense Src expression vectors increases susceptibility to anoikis. In contrast, increasing or decreasing Src expression had no effect on susceptibility to staurosporine-induced apoptosis in attached cells. PD173955, a Src family-specific tyrosine Kinase inhibitor, increases the susceptibility of HT29 cells to anoikis in a dose- and time-dependent manner. Increasing Src expression and activity led to increased phosphorylation of Akt, a mediator of cellular survival pathways, whereas decreasing Src activity led to decreased Akt phosphorylation. In colon tumor cells with high Src activity, the PI3 Kinase inhibitor LY 294002 sensitized cells to anoikis. These results suggest that Src activation may contribute to colon tumor progression and metastasis in part by activating Akt-mediated survival pathways that decrease sensitivity of detached cells to anoikis.
