Ibrutinib treatment improves T cell number and function in CLL patients

PubMed Central

Long, Meixiao; Do, Priscilla; Mundy, Bethany L.; Gordon, Amber; Lehman, Amy M.; Maddocks, Kami J.; Cheney, Carolyn; Jones, Jeffrey A.; Flynn, Joseph M.; Andritsos, Leslie A.; Fraietta, Joseph A.; June, Carl H.; Maus, Marcela V.; Woyach, Jennifer A.; Caligiuri, Michael A.; Johnson, Amy J.

2017-01-01

BACKGROUND. Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton’s tyrosine kinase (BTK) and IL-2–inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies. METHODS. Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated. RESULTS. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4+ T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells. CONCLUSIONS. Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers. TRIAL REGISTRATION. ClinicalTrials.gov NCT01589302 and NCT02029443. Samples described here were collected per OSU-0025. FUNDING. The National Cancer Institute. PMID:28714866

Ibrutinib treatment improves T cell number and function in CLL patients.

PubMed

Long, Meixiao; Beckwith, Kyle; Do, Priscilla; Mundy, Bethany L; Gordon, Amber; Lehman, Amy M; Maddocks, Kami J; Cheney, Carolyn; Jones, Jeffrey A; Flynn, Joseph M; Andritsos, Leslie A; Awan, Farrukh; Fraietta, Joseph A; June, Carl H; Maus, Marcela V; Woyach, Jennifer A; Caligiuri, Michael A; Johnson, Amy J; Muthusamy, Natarajan; Byrd, John C

2017-08-01

Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton's tyrosine kinase (BTK) and IL-2-inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies. Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4+ T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells. Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers. ClinicalTrials.gov NCT01589302 and NCT02029443. Samples described here were collected per OSU-0025. The National Cancer Institute.

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells

PubMed Central

Sela, Meirav; Bogin, Yaron; Beach, Dvora; Oellerich, Thomas; Lehne, Johanna; Smith-Garvin, Jennifer E; Okumura, Mariko; Starosvetsky, Elina; Kosoff, Rachelle; Libman, Evgeny; Koretzky, Gary; Kambayashi, Taku; Urlaub, Henning; Wienands, Jürgen; Chernoff, Jonathan; Yablonski, Deborah

2011-01-01

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1. PMID:21725281

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells.

PubMed

Sela, Meirav; Bogin, Yaron; Beach, Dvora; Oellerich, Thomas; Lehne, Johanna; Smith-Garvin, Jennifer E; Okumura, Mariko; Starosvetsky, Elina; Kosoff, Rachelle; Libman, Evgeny; Koretzky, Gary; Kambayashi, Taku; Urlaub, Henning; Wienands, Jürgen; Chernoff, Jonathan; Yablonski, Deborah

2011-07-01

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1.

Inhibitors of BTK and ITK: state of the new drugs for cancer, autoimmunity and inflammatory diseases.

PubMed

Vargas, L; Hamasy, A; Nore, B F; Smith, C I E

2013-08-01

BTK and ITK are cytoplasmic tyrosine kinases of crucial importance for B and T cell development, with loss-of-function mutations causing X-linked agammaglobulinemia and susceptibility to severe, frequently lethal, Epstein-Barr virus infection, respectively. Over the last few years, considerable efforts have been made in order to develop small-molecule inhibitors for these kinases to treat lymphocyte malignancies, autoimmunity or allergy/hypersensitivity. The rationale is that even if complete lack of BTK or ITK during development causes severe immunodeficiency, inactivation after birth may result in a less severe phenotype. Moreover, therapy can be transient or only partially block the activity of BTK or ITK. Furthermore, a drug-induced B cell deficiency is treatable by gamma globulin substitution therapy. The newly developed BTK inhibitor PCI-32765, recently renamed Ibrutinib, has already entered several clinical trials for various forms of non-Hodgkin lymphoma as well as for multiple myeloma. Experimental animal studies have demonstrated highly promising treatment effects also in autoimmunity. ITK inhibitors are still under the early developmental phase, but it can be expected that such drugs will also become very useful. In this study, we present BTK and ITK with their signalling pathways and review the development of the corresponding inhibitors. © 2013 John Wiley & Sons Ltd.

PET imaging of T cells: Target identification and feasibility assessment.

PubMed

Auberson, Yves P; Briard, Emmanuelle; Rudolph, Bettina; Kaupmann, Klemen; Smith, Paul; Oberhauser, Berndt

2018-06-01

Imaging T cells using positron emission tomography (PET) would be highly useful for diagnosis and monitoring in immunology and oncology patients. There are however no obvious targets that can be used to develop imaging agents for this purpose. We evaluated several potential target proteins with selective expression in T cells, and for which lead molecules were available: PKC , Lck, ZAP70 and Itk. Ultimately, we focused on Itk (interleukin-2-inducible T cell kinase) and identified a tool molecule with properties suitable for in vivo imaging of T cells, (5aR)-5,5-difluoro-5a-methyl-N-(1-((S)-3-(methylsulfonyl)-phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydro-cyclopropa[f]-indazole-3-carboxamide (23). While not having the optimal profile for clinical use, this molecule indicates that it might be possible to develop Itk-selective PET ligands for imaging the distribution of T cells in patients. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Non-receptor tyrosine kinases ITK and BTK negatively regulate mast cell pro-inflammatory responses to lipopolysaccharide

PubMed Central

Huang, Weishan; Morales, J. Luis; Gozivoda, Victor P.; August, Avery

2015-01-01

Background Mast cells are indispensible for LPS-induced septic hypothermia, in which TNF-α plays an essential role to initiate septic responses. ITK and BTK regulate mast cell responses to allergen, but their roles in mast cell responses in LPS-induced sepsis are unclear. Objectives We sought to investigate the roles of ITK and BTK in mast cell responses during LPS-induced septic inflammation. Methods Mice (genetically modified or BMMC-reconstituted Sash) were given LPS to induce septic hypothermia, in the presence or absence of indicated inhibitors. Flow cytometry was used to determine LPS-induced cell influx and TNF-α production in peritoneal cells. Microarray was used for genome-wide gene expression analysis on BMMCs. Quantitative PCR and multiplex were used to determine transcribed and secreted pro-inflammatory cytokines. Microscopy and western blotting were used to determine activation of signal transduction pathways. Results The absence of ITK and BTK leads to exacerbation of LPS-induced septic hypothermia and neutrophil influx. Itk−/−Btk−/− mast cells exhibit hyperactive preformed and LPS-induced TNF-α production, and lead to more severe LPS-induced septic hypothermia when reconstituted into mast cell deficient Sash mice. LPS-induced NF-κB, Akt and p38 activation is enhanced in Itk−/−Btk−/− mast cells, and blockage of PI3K, Akt or p38 downstream MNK1 activation significantly suppresses TNF-α hyper-production and attenuates septic hypothermia. Conclusions ITK and BTK regulate thermal homeostasis during septic response through mast cell function in mice. They share regulatory function downstream of TLR4/LPS in mast cells, through regulating the activation of canonical NF-κB, PI3K/Akt and p38 signaling pathways. PMID:26581914

Quantitative Phosphoproteomics Reveals SLP-76 Dependent Regulation of PAG and Src Family Kinases in T Cells

PubMed Central

Cao, Lulu; Ding, Yiyuan; Hung, Norris; Yu, Kebing; Ritz, Anna; Raphael, Benjamin J.; Salomon, Arthur R.

2012-01-01

The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway. PMID:23071622

Quantitative phosphoproteomics reveals SLP-76 dependent regulation of PAG and Src family kinases in T cells.

PubMed

Cao, Lulu; Ding, Yiyuan; Hung, Norris; Yu, Kebing; Ritz, Anna; Raphael, Benjamin J; Salomon, Arthur R

2012-01-01

The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.

ITK Gene Mutation: Effect on Survival of Children with Severe Hemophagocytic Lymphohistiocytosis.

PubMed

Zheng, Fang; Li, Juan; Zha, Hui; Zhang, Jue; Zhang, Zhiquan; Cheng, Fangjun

2016-11-01

Hemophagocytic lymphohistiocytosis (HLH) is characterized by deadly hyperinflammatory syndrome, but data on severe HLH with multi-organ dysfunction in children are scant. The authors report a retrospective study of 8 cases with severe HLH from a pediatric intensive care unit (PICU) over a 1-y period and found that Epstein barr virus (EBV) -infection was the most common etiology. All patients had genetic analysis, which showed that four patients with EBV -infection had one homozygous mutation, c.985+75G>A (at position chr5:156667232) in exon10 of the ITK gene with poor survival rates. ITK + mutation group had higher percentages of CD3 + CD8 + T cells (36.0 ± 8.4 %) than those in ITK - mutation group (28.8 ± 5.5 %), while they had similar levels of CD3 + CD4 + T cells. ITK + mutation group had lower proportion of CD3 - CD19 + B cells (16.3 ± 2.9 %) and CD16 + CD56 + NK cells (8.4 ± 2.6 %) than ITK - mutation group (29.6 ± 5.1 % and 15.9 ± 9.0 % respectively). Most importantly, patients with EBV infection with c.985+75G>A mutation in ITK had lower survival rates than ITK - mutation group which it may be related with cellular immune dysfunction.

Inhibition of BTK and ITK with Ibrutinib Is Effective in the Prevention of Chronic Graft-versus-Host Disease in Mice

PubMed Central

Nguyen, Hung; Bastian, David; Heinrichs, Jessica; Wu, Yongxia; Liu, Chen; McDonald, Daniel G.; Pidala, Joseph; Yu, Xue-Zhong

2015-01-01

Bruton’s Tyrosine Kinase (BTK) and IL-2 Inducible T-cell Kinase (ITK) are enzymes responsible for the phosphorylation and activation of downstream effectors in the B-cell receptor (BCR) signaling and T cell receptor (TCR) signaling pathways, respectively. Ibrutinib is an FDA-approved potent inhibitor of both BTK and ITK that impairs B-cell and T-cell function. CD4 T cells and B cells are essential for the induction of chronic graft-versus-host disease (cGVHD). We evaluated these targets by testing the ability of Ibrutinib to prevent or ameliorate cGVHD, which is one of the major complications for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). We found that Ibrutinib significantly alleviated cGVHD across four different mouse models, accompanied by increased long-term survival and reduced clinical score. The clinical improvements in Ibrutinib-treated recipients were associated with decreased serum-autoantibodies, costimulatory molecule activation, B-cell proliferation, and glomerulonephritis compared to vehicle controls. Ibrutinib was also able to alleviate the clinical manifestations in acute GVHD (aGVHD), where the recipients were given grafts with or without B cells, suggesting that an inhibitory effect of Ibrutinib on T cells contributes to a reduction in both aGVHD and cGVHD pathogenesis. An effective prophylactic regimen is still lacking to both reduce the incidence and severity of human cGVHD following allo-HSCT. Our study shows that Ibrutinib is an effective prophylaxis against several mouse models of cGVHD with minimal toxicity and could be a promising strategy to combat human cGVHD clinically. PMID:26348529

Inhibition of BTK and ITK with Ibrutinib Is Effective in the Prevention of Chronic Graft-versus-Host Disease in Mice.

PubMed

Schutt, Steven D; Fu, Jianing; Nguyen, Hung; Bastian, David; Heinrichs, Jessica; Wu, Yongxia; Liu, Chen; McDonald, Daniel G; Pidala, Joseph; Yu, Xue-Zhong

2015-01-01

Bruton's Tyrosine Kinase (BTK) and IL-2 Inducible T-cell Kinase (ITK) are enzymes responsible for the phosphorylation and activation of downstream effectors in the B-cell receptor (BCR) signaling and T cell receptor (TCR) signaling pathways, respectively. Ibrutinib is an FDA-approved potent inhibitor of both BTK and ITK that impairs B-cell and T-cell function. CD4 T cells and B cells are essential for the induction of chronic graft-versus-host disease (cGVHD). We evaluated these targets by testing the ability of Ibrutinib to prevent or ameliorate cGVHD, which is one of the major complications for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). We found that Ibrutinib significantly alleviated cGVHD across four different mouse models, accompanied by increased long-term survival and reduced clinical score. The clinical improvements in Ibrutinib-treated recipients were associated with decreased serum-autoantibodies, costimulatory molecule activation, B-cell proliferation, and glomerulonephritis compared to vehicle controls. Ibrutinib was also able to alleviate the clinical manifestations in acute GVHD (aGVHD), where the recipients were given grafts with or without B cells, suggesting that an inhibitory effect of Ibrutinib on T cells contributes to a reduction in both aGVHD and cGVHD pathogenesis. An effective prophylactic regimen is still lacking to both reduce the incidence and severity of human cGVHD following allo-HSCT. Our study shows that Ibrutinib is an effective prophylaxis against several mouse models of cGVHD with minimal toxicity and could be a promising strategy to combat human cGVHD clinically.

An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition.

PubMed

Devkota, Sujan; Joseph, Raji E; Boyken, Scott E; Fulton, D Bruce; Andreotti, Amy H

2017-06-13

Pleckstrin homology (PH) domains are well-known as phospholipid binding modules, yet evidence that PH domain function extends beyond lipid recognition is mounting. In this work, we characterize a protein binding function for the PH domain of interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of nonreceptor tyrosine kinases. Its N-terminal PH domain is a well-characterized lipid binding module that localizes ITK to the membrane via phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) binding. Using a combination of nuclear magnetic resonance spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK, inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP 3 . By clarifying the allosteric role of the ITK PH domain in controlling ITK function, we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.

SimITK: visual programming of the ITK image-processing library within Simulink.

PubMed

Dickinson, Andrew W L; Abolmaesumi, Purang; Gobbi, David G; Mousavi, Parvin

2014-04-01

The Insight Segmentation and Registration Toolkit (ITK) is a software library used for image analysis, visualization, and image-guided surgery applications. ITK is a collection of C++ classes that poses the challenge of a steep learning curve should the user not have appropriate C++ programming experience. To remove the programming complexities and facilitate rapid prototyping, an implementation of ITK within a higher-level visual programming environment is presented: SimITK. ITK functionalities are automatically wrapped into "blocks" within Simulink, the visual programming environment of MATLAB, where these blocks can be connected to form workflows: visual schematics that closely represent the structure of a C++ program. The heavily templated C++ nature of ITK does not facilitate direct interaction between Simulink and ITK; an intermediary is required to convert respective data types and allow intercommunication. As such, a SimITK "Virtual Block" has been developed that serves as a wrapper around an ITK class which is capable of resolving the ITK data types to native Simulink data types. Part of the challenge surrounding this implementation involves automatically capturing and storing the pertinent class information that need to be refined from an initial state prior to being reflected within the final block representation. The primary result from the SimITK wrapping procedure is multiple Simulink block libraries. From these libraries, blocks are selected and interconnected to demonstrate two examples: a 3D segmentation workflow and a 3D multimodal registration workflow. Compared to their pure-code equivalents, the workflows highlight ITK usability through an alternative visual interpretation of the code that abstracts away potentially confusing technicalities.

Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation

PubMed Central

Alcázar, Isabela; Marqués, Miriam; Kumar, Amit; Hirsch, Emilio; Wymann, Matthias; Carrera, Ana C.; Barber, Domingo F.

2007-01-01

Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation. PMID:17998387

The Design of SimpleITK.

PubMed

Lowekamp, Bradley C; Chen, David T; Ibáñez, Luis; Blezek, Daniel

2013-01-01

SimpleITK is a new interface to the Insight Segmentation and Registration Toolkit (ITK) designed to facilitate rapid prototyping, education and scientific activities via high level programming languages. ITK is a templated C++ library of image processing algorithms and frameworks for biomedical and other applications, and it was designed to be generic, flexible and extensible. Initially, ITK provided a direct wrapping interface to languages such as Python and Tcl through the WrapITK system. Unlike WrapITK, which exposed ITK's complex templated interface, SimpleITK was designed to provide an easy to use and simplified interface to ITK's algorithms. It includes procedural methods, hides ITK's demand driven pipeline, and provides a template-less layer. Also SimpleITK provides practical conveniences such as binary distribution packages and overloaded operators. Our user-friendly design goals dictated a departure from the direct interface wrapping approach of WrapITK, toward a new facade class structure that only exposes the required functionality, hiding ITK's extensive template use. Internally SimpleITK utilizes a manual description of each filter with code-generation and advanced C++ meta-programming to provide the higher-level interface, bringing the capabilities of ITK to a wider audience. SimpleITK is licensed as open source software library under the Apache License Version 2.0 and more information about downloading it can be found at http://www.simpleitk.org.

The Design of SimpleITK

PubMed Central

Lowekamp, Bradley C.; Chen, David T.; Ibáñez, Luis; Blezek, Daniel

2013-01-01

SimpleITK is a new interface to the Insight Segmentation and Registration Toolkit (ITK) designed to facilitate rapid prototyping, education and scientific activities via high level programming languages. ITK is a templated C++ library of image processing algorithms and frameworks for biomedical and other applications, and it was designed to be generic, flexible and extensible. Initially, ITK provided a direct wrapping interface to languages such as Python and Tcl through the WrapITK system. Unlike WrapITK, which exposed ITK's complex templated interface, SimpleITK was designed to provide an easy to use and simplified interface to ITK's algorithms. It includes procedural methods, hides ITK's demand driven pipeline, and provides a template-less layer. Also SimpleITK provides practical conveniences such as binary distribution packages and overloaded operators. Our user-friendly design goals dictated a departure from the direct interface wrapping approach of WrapITK, toward a new facade class structure that only exposes the required functionality, hiding ITK's extensive template use. Internally SimpleITK utilizes a manual description of each filter with code-generation and advanced C++ meta-programming to provide the higher-level interface, bringing the capabilities of ITK to a wider audience. SimpleITK is licensed as open source software library under the Apache License Version 2.0 and more information about downloading it can be found at http://www.simpleitk.org. PMID:24416015

SimITK: rapid ITK prototyping using the Simulink visual programming environment

NASA Astrophysics Data System (ADS)

Dickinson, A. W. L.; Mousavi, P.; Gobbi, D. G.; Abolmaesumi, P.

2011-03-01

The Insight Segmentation and Registration Toolkit (ITK) is a long-established, software package used for image analysis, visualization, and image-guided surgery applications. This package is a collection of C++ libraries, that can pose usability problems for users without C++ programming experience. To bridge the gap between the programming complexities and the required learning curve of ITK, we present a higher-level visual programming environment that represents ITK methods and classes by wrapping them into "blocks" within MATLAB's visual programming environment, Simulink. These blocks can be connected to form workflows: visual schematics that closely represent the structure of a C++ program. Due to the heavily C++ templated nature of ITK, direct interaction between Simulink and ITK requires an intermediary to convert their respective datatypes and allow intercommunication. We have developed a "Virtual Block" that serves as an intermediate wrapper around the ITK class and is responsible for resolving the templated datatypes used by ITK to native types used by Simulink. Presently, the wrapping procedure for SimITK is semi-automatic in that it requires XML descriptions of the ITK classes as a starting point, as this data is used to create all other necessary integration files. The generation of all source code and object code from the XML is done automatically by a CMake build script that yields Simulink blocks as the final result. An example 3D segmentation workflow using cranial-CT data as well as a 3D MR-to-CT registration workflow are presented as a proof-of-concept.

Ibrutinib Inhibits ERBB Receptor Tyrosine Kinases and HER2-Amplified Breast Cancer Cell Growth.

PubMed

Chen, Jun; Kinoshita, Taisei; Sukbuntherng, Juthamas; Chang, Betty Y; Elias, Laurence

2016-12-01

Ibrutinib is a potent, small-molecule Bruton tyrosine kinase (BTK) inhibitor developed for the treatment of B-cell malignancies. Ibrutinib covalently binds to Cys481 in the ATP-binding domain of BTK. This cysteine residue is conserved among 9 other tyrosine kinases, including HER2 and EGFR, which can be targeted. Screening large panels of cell lines demonstrated that ibrutinib was growth inhibitory against some solid tumor cells, including those inhibited by other HER2/EGFR inhibitors. Among sensitive cell lines, breast cancer lines with HER2 overexpression were most potently inhibited by ibrutinib (<100 nmol/L); in addition, the IC 50 s were lower than that of lapatinib and dacomitinib. Inhibition of cell growth by ibrutinib coincided with downregulation of phosphorylation on HER2 and EGFR and their downstream targets, AKT and ERK. Irreversible inhibition of HER2 and EGFR in breast cancer cells was established after 30-minute incubation above 100 nmol/L or following 2-hour incubation at lower concentrations. Furthermore, ibrutinib inhibited recombinant HER2 and EGFR activity that was resistant to dialysis and rapid dilution, suggesting an irreversible interaction. The dual activity toward TEC family (BTK and ITK) and ERBB family kinases was unique to ibrutinib, as ERBB inhibitors do not inhibit or covalently bind BTK or ITK. Xenograft studies with HER2 + MDA-MB-453 and BT-474 cells in mice in conjunction with determination of pharmacokinetics demonstrated significant exposure-dependent inhibition of growth and key signaling molecules at levels that are clinically achievable. Ibrutinib's unique dual spectrum of activity against both TEC family and ERBB kinases suggests broader applications of ibrutinib in oncology. Mol Cancer Ther; 15(12); 2835-44. ©2016 AACR. ©2016 American Association for Cancer Research.

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

PubMed Central

Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

2014-01-01

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T-Cell Receptor and SLP-76 Complex.

PubMed

Belmont, Judson; Gu, Tao; Mudd, Ashley; Salomon, Arthur R

2017-08-04

Phospholipase C gamma 1 (PLC-γ1) occupies a critically important position in the T-cell signaling pathway. While its functions as a regulator of both Ca 2+ signaling and PKC-family kinases are well characterized, PLC-γ1's role in the regulation of early T-cell receptor signaling events is incompletely understood. Activation of the T-cell receptor leads to the formation of a signalosome complex between SLP-76, LAT, PLC-γ1, Itk, and Vav1. Recent studies have revealed the existence of both positive and negative feedback pathways from SLP-76 to the apical kinase in the pathway, Lck. To determine if PLC-γ1 contributes to the regulation of these feedback networks, we performed a quantitative phosphoproteomic analysis of PLC-γ1-deficient T cells. These data revealed a previously unappreciated role for PLC-γ1 in the positive regulation of Zap-70 and T-cell receptor tyrosine phosphorylation. Conversely, PLC-γ1 negatively regulated the phosphorylation of SLP-76-associated proteins, including previously established Lck substrate phosphorylation sites within this complex. While the positive and negative regulatory phosphorylation sites on Lck were largely unchanged, Tyr 192 phosphorylation was elevated in Jgamma1. The data supports a model wherein Lck's targeting, but not its kinase activity, is altered by PLC-γ1, possibly through Lck Tyr 192 phosphorylation and increased association of the kinase with protein scaffolds SLP-76 and TSAd.

Protein kinase D2 is a digital amplifier of T cell receptor–stimulated diacylglycerol signaling in naïve CD8+ T cells

PubMed Central

Navarro, María N.; Feijoo-Carnero, Carmen; Arandilla, Alba Gonzalez; Trost, Matthias; Cantrell, Doreen A.

2016-01-01

Protein kinase D2 (PKD2) is a serine and threonine kinase that is activated in T cells by diacylglycerol and protein kinase C in response to stimulation of the T cell receptor (TCR) by antigen. We quantified the activation of PKD2 at the single-cell level and found that this kinase acts as a sensitive digital amplifier of TCR engagement, enabling CD8+ T cells to match the production of inflammatory cytokines to the quality and quantity of TCR ligands. There was a digital response pattern of PKD2 activation in response to TCR engagement, such that increasing the concentration and potency of TCR ligands increased the number of cells that exhibited activated PKD2. However, for each cell that responded to TCR stimulation, the entire cellular pool of PKD2 (~400,000 molecules) was activated. Moreover, PKD2 acted as an amplification checkpoint for antigen-stimulated digital cytokine responses and translated the differential strength of TCR signaling to determine the number of naïve CD8+ T cells that became effector cells. Together, these results provide insights into PKD family kinases and how they act digitally to amplify signaling networks controlled by the TCR. PMID:25336615

Systematic review of infectious events with the Bruton tyrosine kinase inhibitor ibrutinib in the treatment of hematologic malignancies.

PubMed

Tillman, Benjamin F; Pauff, James M; Satyanarayana, Gowri; Talbott, Mahsa; Warner, Jeremy L

2018-04-01

Ibrutinib is an irreversible inhibitor of Bruton tyrosine kinase (BTK) in B lymphocytes as well as other kinases including interleukin-2-inducible T-cell kinase (ITK) in CD4+ Th2 regulatory T cells. Increased infections have been observed in patients taking ibrutinib. The overall incidence has not been systematically evaluated. The published literature and conference abstracts of prospective clinical trials using ibrutinib in hematologic malignancies were identified and reviewed using PubMed, Google Scholar, and HemOnc.org per PRISMA guidelines. Infectious events with a focus on pneumonia were collated per the Common Terminology Criteria for Adverse Events Version 4.03 grading. Infectious complications are common, occurring in 56% of patients taking single-agent ibrutinib and 52% of those on combination therapy. Approximately one in 5 patients developed pneumonia, which was the major contributor to a 2% rate of death from infections. Many of the cases of pneumonia were due to opportunistic pathogens. Ibrutinib use requires prudent consideration of the impacts on host immunity. We identified a high rate of serious adverse infectious events within prospective clinical trials. Data suggest a role of both BTK and ITK inhibition for the increased events. There was considerable variability in the reporting of adverse events between trials, journals, and conference reports. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Simultaneous inhibition of pan-phosphatidylinositol-3-kinases and MEK as a potential therapeutic strategy in peripheral T-cell lymphomas.

PubMed

Martín-Sánchez, Esperanza; Rodríguez-Pinilla, Socorro M; Sánchez-Beato, Margarita; Lombardía, Luis; Domínguez-González, Beatriz; Romero, Diana; Odqvist, Lina; García-Sanz, Pablo; Wozniak, Magdalena B; Kurz, Guido; Blanco-Aparicio, Carmen; Mollejo, Manuela; Alves, F Javier; Menárguez, Javier; González-Palacios, Fernando; Rodríguez-Peralto, José Luis; Ortiz-Romero, Pablo L; García, Juan F; Bischoff, James R; Piris, Miguel A

2013-01-01

Peripheral T-cell lymphomas are very aggressive hematologic malignancies for which there is no targeted therapy. New, rational approaches are necessary to improve the very poor outcome in these patients. Phosphatidylinositol-3-kinase is one of the most important pathways in cell survival and proliferation. We hypothesized that phosphatidylinositol-3-kinase inhibitors could be rationally selected drugs for treating peripheral T-cell lymphomas. Several phosphatidylinositol-3-kinase isoforms were inhibited genetically (using small interfering RNA) and pharmacologically (with CAL-101 and GDC-0941 compounds) in a panel of six peripheral and cutaneous T-cell lymphoma cell lines. Cell viability was measured by intracellular ATP content; apoptosis and cell cycle changes were checked by flow cytometry. Pharmacodynamic biomarkers were assessed by western blot. The PIK3CD gene, which encodes the δ isoform of phosphatidylinositol-3-kinase, was overexpressed in cell lines and primary samples, and correlated with survival pathways. However, neither genetic nor specific pharmacological inhibition of phosphatidylinositol-3-kinase δ affected cell survival. In contrast, the pan-phosphatidylinositol-3-kinase inhibitor GDC-0941 arrested all T-cell lymphoma cell lines in the G1 phase and induced apoptosis in a subset of them. We identified phospho-GSK3β and phospho-p70S6K as potential biomarkers of phosphatidylinositol-3-kinase inhibitors. Interestingly, an increase in ERK phosphorylation was observed in some GDC -0941-treated T-cell lymphoma cell lines, suggesting the presence of a combination of phosphatidylinositol-3-kinase and MEK inhibitors. A highly synergistic effect was found between the two inhibitors, with the combination enhancing cell cycle arrest at G0/G1 in all T-cell lymphoma cell lines, and reducing cell viability in primary tumor T cells ex vivo. These results suggest that the combined treatment of pan-phosphatidylinositol-3-kinase + MEK inhibitors could be more

Bruton's tyrosine kinase (BTK) as a promising target in solid tumors.

PubMed

Molina-Cerrillo, J; Alonso-Gordoa, T; Gajate, P; Grande, E

2017-07-01

Bruton's tyrosine kinase (BTK) is a non-receptor intracellular kinase that belongs to the TEC-family tyrosine kinases together with bone marrow-expressed kinase (BMX), redundant-resting lymphocyte kinase (RLK), and IL-2 inducible T-Cell kinase (ITK). All these proteins play a key role in the intracellular signaling of both B and T lymphocytes. Recently, some preclinical data have demonstrated that BTK is present in certain tumor subtypes and in other relevant cells that are contributing to the tumor microenvironment such as dendritic cells, macrophages, myeloid derived suppressor cells and endothelial cells. Ibrutinib (PCI-32765) is an orally available small molecule that acts as an inhibitor of the BTK and is approved for the treatment of patients with some hematological malignancies. It has been suggested that ibrutinib may also have a potential antitumor activity in solid neoplasms. In this sense, ibrutinib has the ability to revert polarization of TCD4+ to Th1 lymphocytes to increase the cytotoxic ability of T CD8+ and to regulate tumor-induced immune tolerance by acting over tumor infiltrating cells activity and immunosuppressive cytokines release. Furthermore, based on its molecular activity and safety, ibrutinib has been considered as a partner for treatment combination with PI3K/AKT/mTOR inhibitors or with immune-checkpoint inhibitors, inhibiting immunosuppressive signals from the tumor microenvironment, and overcoming the immune resistance to current anti-PD1/PDL1 immunotherapeutic drugs by the CXCR4/CXCL2 pathway regulation. Currently, a broad range of different studies are evaluating the activity of ibrutinib either as single agent or in combination in patients with solid tumors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Deregulated activation of oncoprotein kinase Tpl2/Cot in HTLV-I-transformed T cells.

PubMed

Babu, Geetha; Waterfield, Michael; Chang, Mikyoung; Wu, Xuefeng; Sun, Shao-Cong

2006-05-19

Protein kinase Tpl2/Cot is encoded by a protooncogene that is cis-activated by retroviral insertion in murine T cell lymphomas. It has remained unclear whether this oncoprotein kinase is mutated or post-translationally activated in human cancer cells. We have shown here that Tpl2/Cot is constitutively activated in human leukemia cell lines transformed by the human T cell leukemia virus type I (HTLV-I). The kinase activity of Tpl2/Cot is normally suppressed through its physical interaction with an inhibitor, the NF-kappaB1 precursor protein p105. Interestingly, a large pool of Tpl2/Cot is liberated from p105 and exhibits constitutive kinase activity in HTLV-I-transformed T cells. In contrast to its labile property in normal cells, the pathologically activated Tpl2/Cot is remarkably stable. Further, whereas the physiological activation of Tpl2/Cot involves its long isoform, the HTLV-activated Tpl2/Cot is predominantly the short isoform. We have also shown that the HTLV-I-encoded Tax protein is able to activate Tpl2/Cot in transfected cells. Finally, Tpl2/Cot participates in the activation of NF-kappaB by Tax. These findings indicate that deregulated activation of Tpl2/Cot may occur in human cancer cells.

The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trinks, Cecilia, E-mail: Cecilia.trinks@liu.se; Severinsson, Emelie A., E-mail: Emelie.severinsson@liu.se; Holmlund, Birgitta, E-mail: Birgitta.holmlund@lio.se

2011-07-08

Highlights: {yields} Canertinib induces caspase-mediated apoptosis in T-cell leukemia cells in vitro. {yields} Canertinib mediates activation of the intrinsic apoptotic pathway. {yields} Canertinib induces apoptosis in an ErbB receptor independent manner. {yields} Lymphocyte specific proteins as well as survival kinases are inhibited. {yields} Canertinib may act as a multi-kinase inhibiting drug in human T-cell malignancies. -- Abstract: Canertinib is a novel ErbB-receptor inhibitor currently in clinical development for the treatment of solid tumors overexpressing ErbB-receptors. We have recently demonstrated that canertinib displays anti-proliferative and pro-apoptotic effects in human myeloid leukemia cells devoid of ErbB-receptors. The mechanism mediating these effects aremore » however unknown. In this study, we show that canertinib is able to act as a multi-kinase inhibitor by inhibition of several intracellular kinases involved in T-cell signaling such as Akt, Erk1/2 and Zap-70, and reduced Lck protein expression in the human T-cell leukemia cell line Jurkat. Treatment with canertinib at a concentration of 2 {mu}M caused accumulation of Jurkat cells in the G{sub 1} cell cycle phase and increased doses induced apoptosis in a time-dependent manner. Apoptotic signs of treated cells were detected by Annexin V staining and cleavage of PARP, caspase-3, -8, -9, -10 and Bid. A subset of the pro-apoptotic signals mediated by canertinib could be significantly reduced by specific caspase inhibitors. Taken together, these results demonstrate the dual ability of canertinib to downregulate important signaling pathways and to activate caspase-mediated intrinsic apoptosis pathway in human T-cell leukemia cells.« less

MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells.

PubMed

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.

The Non-Classical MAP Kinase ERK3 Controls T Cell Activation

PubMed Central

Mathien, Simon; Rousseau, Justine; Thébault, Paméla; Daudelin, Jean-François; Rooney, Julie; Turgeon, Benjamin; Beauchamp, Claudine; Meloche, Sylvain; Labrecque, Nathalie

2014-01-01

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4+ and CD8+ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation. PMID:24475167

The non-classical MAP kinase ERK3 controls T cell activation.

PubMed

Marquis, Miriam; Boulet, Salix; Mathien, Simon; Rousseau, Justine; Thébault, Paméla; Daudelin, Jean-François; Rooney, Julie; Turgeon, Benjamin; Beauchamp, Claudine; Meloche, Sylvain; Labrecque, Nathalie

2014-01-01

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.

An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor

PubMed Central

Shah, Neel H; Wang, Qi; Yan, Qingrong; Karandur, Deepti; Kadlecek, Theresa A; Fallahee, Ian R; Russ, William P; Ranganathan, Rama; Weiss, Arthur; Kuriyan, John

2016-01-01

The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens. DOI: http://dx.doi.org/10.7554/eLife.20105.001 PMID:27700984

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.

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 providesmore » 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.« less

Interaction between Sam68 and Src family tyrosine kinases, Fyn and Lck, in T cell receptor signaling.

PubMed

Fusaki, N; Iwamatsu, A; Iwashima, M; Fujisawa, J i

1997-03-07

The Src family protein-tyrosine kinase, Fyn, is associated with the T cell receptor (TCR) and plays an important role in TCR-mediated signaling. We found that a human T cell leukemia virus type 1-infected T cell line, Hayai, overexpressed Fyn. To identify the molecules downstream of Fyn, we analyzed the tyrosine phosphorylation of cellular proteins in the cells. In Hayai, a 68-kDa protein was constitutively tyrosine-phosphorylated. The 68-kDa protein was coimmunoprecipitated with various signaling proteins such as phospholipase C gamma1, the phosphatidylinositol 3-kinase p85 subunit, Grb2, SHP-1, Cbl, and Jak3, implying that the protein might function as an adapter. Purification and microsequencing of this protein revealed that it was the RNA-binding protein, Sam68 (Src associated in mitosis, 68 kDa). Sam68 was associated with the Src homology 2 and 3 domains of Fyn and also those of another Src family kinase, Lck. CD3 cross-linking induced tyrosine phosphorylation of Sam68 in uninfected T cells. These data suggest that Sam68 participates in the signal transduction pathway downstream of TCR-coupled Src family kinases Fyn and Lck in lymphocytes, that is not only in the mitotic pathway downstream of c-Src in fibroblasts.

Tpl2 kinase regulates T cell interferon-γ production and host resistance to Toxoplasma gondii

PubMed Central

Watford, Wendy T.; Hissong, Bruce D.; Durant, Lydia R.; Yamane, Hidehiro; Muul, Linda M.; Kanno, Yuka; Tato, Cristina M.; Ramos, Haydeé L.; Berger, Alan E.; Mielke, Lisa; Pesu, Marko; Solomon, Benjamin; Frucht, David M.; Paul, William E.; Sher, Alan; Jankovic, Dragana; Tsichlis, Philip N.; O'Shea, John J.

2008-01-01

Tpl2 (Tumor progression locus 2), also known as Cot/MAP3K8, is a hematopoietically expressed serine-threonine kinase. Tpl2 is known to have critical functions in innate immunity in regulating tumor necrosis factor–α, Toll-like receptor, and G protein–coupled receptor signaling; however, our understanding of its physiological role in T cells is limited. We investigated the potential roles of Tpl2 in T cells and found that it was induced by interleukin-12 in human and mouse T cells in a Stat4-dependent manner. Deficiency of Tpl2 was associated with impaired interferon (IFN)-γ production. Accordingly, Tpl2−/− mice had impaired host defense against Toxoplasma gondii with reduced parasite clearance and decreased IFN-γ production. Furthermore, reconstitution of Rag2−/− mice with Tpl2-deficient T cells followed by T. gondii infection recapitulated the IFN-γ defect seen in the Tpl2-deficient mice, confirming a T cell–intrinsic defect. CD4+ T cells isolated from Tpl2−/− mice showed poor induction of T-bet and failure to up-regulate Stat4 protein, which is associated with impaired TCR-dependent extracellular signal-regulated kinase activation. These data underscore the role of Tpl2 as a regulator of T helper cell lineage decisions and demonstrate that Tpl2 has an important functional role in the regulation of Th1 responses. PMID:19001140

Protein kinase Cδ oxidation contributes to ERK inactivation in lupus T cells.

PubMed

Gorelik, Gabriela J; Yarlagadda, Sushma; Patel, Dipak R; Richardson, Bruce C

2012-09-01

CD4+ T cells from patients with active lupus have impaired ERK pathway signaling that decreases DNA methyltransferase expression, resulting in DNA demethylation, overexpression of immune genes, and autoimmunity. The ERK pathway defect is due to impaired phosphorylation of T(505) in the protein kinase Cδ (PKCδ) activation loop. However, the mechanisms that prevent PKCδ T(505) phosphorylation in lupus T cells are unknown. Others have reported that oxidative modifications, and nitration in particular, of T cells as well as serum proteins correlate with lupus disease activity. We undertook this study to test our hypothesis that nitration inactivates PKCδ, contributing to impaired ERK pathway signaling in lupus T cells. CD4+ T cells were purified from lupus patients and controls and then stimulated with phorbol myristate acetate (PMA). Signaling protein levels, nitration, and phosphorylation were quantitated by immunoprecipitation and immunoblotting of T cell lysates. Transfections were performed by electroporation. Treating CD4+ T cells with peroxynitrite nitrated PKCδ, preventing PKCδ T(505) phosphorylation and inhibiting ERK pathway signaling similar to that observed in lupus T cells. Patients with active lupus had higher nitrated T cell PKCδ levels than did controls, which correlated directly with disease activity, and antinitrotyrosine immunoprecipitations demonstrated that nitrated PKCδ, but not unmodified PKCδ, was refractory to PMA-stimulated T(505) phosphorylation, similar to PKCδ in peroxynitrite-treated cells. Oxidative stress causes PKCδ nitration, which prevents its phosphorylation and contributes to the decreased ERK signaling in lupus T cells. These results identify PKCδ as a link between oxidative stress and the T cell epigenetic modifications in lupus. Copyright © 2012 by the American College of Rheumatology.

Investigating small molecules to inhibit germinal center kinase-like kinase (GLK/MAP4K3) upstream of PKCθ phosphorylation: Potential therapy to modulate T cell dependent immunity.

PubMed

May-Dracka, Tricia L; Arduini, Robert; Bertolotti-Ciarlet, Andrea; Bhisetti, Govinda; Brickelmaier, Margot; Cahir-McFarland, Ellen; Enyedy, Istvan; Fontenot, Jason D; Hesson, Thomas; Little, Kevin; Lyssikatos, Joe; Marcotte, Douglas; McKee, Timothy; Murugan, Paramasivam; Patterson, Thomas; Peng, Hairuo; Rushe, Mia; Silvian, Laura; Spilker, Kerri; Wu, Ping; Xin, Zhili; Burkly, Linda C

2018-06-01

Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines. Copyright © 2018 Elsevier Ltd. All rights reserved.

Myeloid-derived suppressor cells express Bruton’s tyrosine kinase and can be depleted in tumor bearing hosts by ibrutinib treatment

PubMed Central

Stiff, Andrew; Trikha, Prashant; Wesolowski, Robert; Kendra, Kari; Hsu, Vincent; Uppati, Sarvani; McMichael, Elizabeth; Duggan, Megan; Campbell, Amanda; Keller, Karen; Landi, Ian; Zhong, Yiming; Dubovsky, Jason; Howard, John Harrison; Yu, Lianbo; Harrington, Bonnie; Old, Matthew; Reiff, Sean; Mace, Thomas; Tridandapani, Susheela; Muthusamy, Natarajan; Caligiuri, Michael A.; Byrd, John C.; Carson, William E.

2016-01-01

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells that expand in tumor bearing hosts in response to soluble factors produced by tumor and stromal cells. MDSC expansion has been linked to loss of immune effector cell function and reduced efficacy of immune-based cancer therapies, highlighting the MDSC population as an attractive therapeutic target. Ibrutinib, an irreversible inhibitor of Bruton’s tyrosine kinase (BTK) and IL2-inducible T-cell kinase (ITK), is in clinical use for the treatment of B cell malignancies. Here, we report that BTK is expressed by murine and human MDSCs, and that ibrutinib is able to inhibit BTK phosphorylation in these cells. Treatment of MDSCs with ibrutinib significantly impaired nitric oxide production and cell migration. In addition, ibrutinib inhibited in vitro generation of human MDSCs and reduced mRNA expression of indolamine 2,3-dioxygenase, an immunosuppressive factor. Treatment of mice bearing EMT6 mammary tumors with ibrutinib resulted in reduced frequency of MDSCs in both the spleen and tumor. Ibrutinib treatment also resulted in a significant reduction of MDSCs in wildtype mice bearing B16F10 melanoma tumors, but not in X-linked immunodeficiency mice (XID) harboring a BTK mutation, suggesting that BTK inhibition plays an important role in the observed reduction of MDSCs in vivo. Finally, ibrutinib significantly enhanced the efficacy of anti-PD-L1 (CD274) therapy in a murine breast cancer model. Together, these results demonstrate that ibrutinib modulates MDSC function and generation, revealing a potential strategy for enhancing immune-based therapies in solid malignancies. PMID:26880800

Human T-Cell Leukemia Virus Type 1 Tax Induction of NF-κB Involves Activation of the IκB Kinase α (IKKα) and IKKβ Cellular Kinases

PubMed Central

Geleziunas, Romas; Ferrell, Sharon; Lin, Xin; Mu, Yajun; Cunningham, Emmett T.; Grant, Mark; Connelly, Margery A.; Hambor, John E.; Marcu, Kenneth B.; Greene, Warner C.

1998-01-01

Tax corresponds to a 40-kDa transforming protein from the pathogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) that activates nuclear expression of the NF-κB/Rel family of transcription factors by an unknown mechanism. Tax expression promotes N-terminal phosphorylation and degradation of IκBα, a principal cytoplasmic inhibitor of NF-κB. Our studies now demonstrate that HTLV-1 Tax activates the recently identified cellular kinases IκB kinase α (IKKα) and IKKβ, which normally phosphorylate IκBα on both of its N-terminal regulatory serines in response to tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1) stimulation. In contrast, a mutant of Tax termed M22, which does not induce NF-κB, fails to activate either IKKα or IKKβ. Furthermore, endogenous IKK enzymatic activity was significantly elevated in HTLV-1-infected and Tax-expressing T-cell lines. Transfection of kinase-deficient mutants of IKKα and IKKβ into either human Jurkat T or 293 cells also inhibits NF-κB-dependent reporter gene expression induced by Tax. Similarly, a kinase-deficient mutant of NIK (NF-κB-inducing kinase), which represents an upstream kinase in the TNF-α and IL-1 signaling pathways leading to IKKα and IKKβ activation, blocks Tax induction of NF-κB. However, plasma membrane-proximal elements in these proinflammatory cytokine pathways are apparently not involved since dominant negative mutants of the TRAF2 and TRAF6 adaptors, which effectively block signaling through the cytoplasmic tails of the TNF-α and IL-1 receptors, respectively, do not inhibit Tax induction of NF-κB. Together, these studies demonstrate that HTLV-1 Tax exploits a distal part of the proinflammatory cytokine signaling cascade leading to induction of NF-κB. The pathological alteration of this cytokine pathway leading to NF-κB activation by Tax may play a central role in HTLV-1-mediated transformation of human T cells, clinically manifested as the adult T-cell leukemia. PMID

Tax-Independent Constitutive IκB Kinase Activation in Adult T-Cell Leukemia Cells1

PubMed Central

Hironaka, Noriko; Mochida, Kanako; Mori, Naoki; Maeda, Michiyuki; Yamamoto, Naoki; Yamaoka, Shoji

2004-01-01

Abstract Adult T-cell leukemia (ATL) is a fatal T-cell malignancy that arises long after infection with human T-cell leukemia virus type I (HTLV-I). We reported previously that nuclear factor-κB (NF-κB) was constitutively activated in ATL cells, although expression of the viral proteins was barely detectable, including Tax, which was known to persistently activate NF-κB. Here we demonstrate that ATL cells that do not express detectable Tax protein exhibit constitutive IκB kinase (IKK) activity. Transfection studies revealed that a dominant-negative form of IKK1, and not of IKK2 or NF-κB essential modulator (NEMO), suppressed constitutive NFκB activity in ATL cells. This IKK activity was accompanied by elevated expression of p52, suggesting that the recently described noncanonical pathway of NF-κB activation operates in ATL cells. We finally show that specific inhibition of NF-κB by a super-repressor form of IκBα (SR-IκBα) in HTLV-I-infected T cells results in cell death regardless of Tax expression, providing definitive evidence of an essential role for NF-κB in the survival of ATL cells. In conclusion, the IKK complex is constitutively activated in ATL cells through a cellular mechanism distinct from that of Tax-mediated IKK activation. Further elucidation of this cellular mechanism should contribute to establishing a rationale for treatment of ATL. PMID:15153339

T-18, a stemonamide synthetic intermediate inhibits Pim kinase activity and induces cell apoptosis, acting as a potent anticancer drug.

PubMed

Wang, Zhen; Li, Xing-Min; Shang, Kun; Zhang, Peng; Wang, Chao-Fu; Xin, Yu-Hu; Zhou, Lu; Li, Ying-Yi

2013-03-01

Pim-3 kinase has been shown to be aberrantly expressed in premalignant and malignant lesions of endoderm-derived organs such as the liver, pancreas, colon and stomach. Pim-3 kinase inactivates the Bad protein, a proapoptotic molecule, and improves the expression of Bcl-xL, an antiapoptotic molecule, to promote cell proliferation. Thus, blocking Pim-3 kinase activity may be a new strategy for the treatment of pancreatic cancer. In this study, we screened low molecular compounds and observed that the stemonamide synthetic intermediate, T-18, potently inhibited Pim kinase activity. Moreover, T-18 inhibited the proliferation of human pancreatic, as well as that of hepatocellular and colon cancer cells in vitro. It also induced the apoptosis of human pancreatic carcinoma cells in vitro by decreasing the levels of phospho-Ser112-Bad; the levels of Pim-3 kinase and total Bad protein were not altered. Furthermore, T-18 inhibited the growth of human pancreatic cancer cells in nude mice without apparent adverse effects when the tumor was palpable. These observations indicate that stemonamide synthetic intermediates may be novel drugs for the treatment of gastrointestinal cancers, particularly pancreatic cancer.

Myeloid-Derived Suppressor Cells Express Bruton's Tyrosine Kinase and Can Be Depleted in Tumor-Bearing Hosts by Ibrutinib Treatment.

PubMed

Stiff, Andrew; Trikha, Prashant; Wesolowski, Robert; Kendra, Kari; Hsu, Vincent; Uppati, Sarvani; McMichael, Elizabeth; Duggan, Megan; Campbell, Amanda; Keller, Karen; Landi, Ian; Zhong, Yiming; Dubovsky, Jason; Howard, John Harrison; Yu, Lianbo; Harrington, Bonnie; Old, Matthew; Reiff, Sean; Mace, Thomas; Tridandapani, Susheela; Muthusamy, Natarajan; Caligiuri, Michael A; Byrd, John C; Carson, William E

2016-04-15

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immature myeloid cells that expand in tumor-bearing hosts in response to soluble factors produced by tumor and stromal cells. MDSC expansion has been linked to loss of immune effector cell function and reduced efficacy of immune-based cancer therapies, highlighting the MDSC population as an attractive therapeutic target. Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and IL2-inducible T-cell kinase (ITK), is in clinical use for the treatment of B-cell malignancies. Here, we report that BTK is expressed by murine and human MDSCs, and that ibrutinib is able to inhibit BTK phosphorylation in these cells. Treatment of MDSCs with ibrutinib significantly impaired nitric oxide production and cell migration. In addition, ibrutinib inhibited in vitro generation of human MDSCs and reduced mRNA expression of indolamine 2,3-dioxygenase, an immunosuppressive factor. Treatment of mice bearing EMT6 mammary tumors with ibrutinib resulted in reduced frequency of MDSCs in both the spleen and tumor. Ibrutinib treatment also resulted in a significant reduction of MDSCs in wild-type mice bearing B16F10 melanoma tumors, but not in X-linked immunodeficiency mice (XID) harboring a BTK mutation, suggesting that BTK inhibition plays an important role in the observed reduction of MDSCs in vivo Finally, ibrutinib significantly enhanced the efficacy of anti-PD-L1 (CD274) therapy in a murine breast cancer model. Together, these results demonstrate that ibrutinib modulates MDSC function and generation, revealing a potential strategy for enhancing immune-based therapies in solid malignancies. Cancer Res; 76(8); 2125-36. ©2016 AACR. ©2016 American Association for Cancer Research.

The C-type lectin OCILRP2 costimulates EL4 T cell activation via the DAP12-Raf-MAP kinase pathway.

PubMed

Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

2014-01-01

OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation.

T cell receptor–induced phosphoinositide-3-kinase p110δ activity is required for T cell localization to antigenic tissue in mice

PubMed Central

Jarmin, Sarah J.; David, Rachel; Ma, Liang; Chai, Jan-Guo; Dewchand, Hamlata; Takesono, Aya; Ridley, Anne J.; Okkenhaug, Klaus; Marelli-Berg, Federica M.

2008-01-01

The establishment of T cell–mediated inflammation requires the migration of primed T lymphocytes from the blood stream and their retention in antigenic sites. While naive T lymphocyte recirculation in the lymph and blood is constitutively regulated and occurs in the absence of inflammation, the recruitment of primed T cells to nonlymphoid tissue and their retention at the site are enhanced by various inflammatory signals, including TCR engagement by antigen-displaying endothelium and resident antigen-presenting cells. In this study, we investigated whether signals downstream of TCR ligation mediated by the phosphoinositide-3-kinase (PI3K) subunit p110δ contributed to the regulation of these events. T lymphocytes from mice expressing catalytically inactive p110δ displayed normal constitutive trafficking and migratory responses to nonspecific stimuli. However, these cells lost susceptibility to TCR-induced migration and failed to localize efficiently to antigenic tissue. Importantly, we showed that antigen-induced T cell trafficking and subsequent inflammation was abrogated by selective pharmacological inhibition of PI3K p110δ activity. These observations suggest that pharmacological targeting of p110δ activity is a viable strategy for the therapy of T cell–mediated pathology. PMID:18259608

The kinases MEKK2 and MEKK3 regulate transforming growth factor-β-mediated helper T cell differentiation.

PubMed

Chang, Xing; Liu, Fang; Wang, Xiaofang; Lin, Aiping; Zhao, Hongyu; Su, Bing

2011-02-25

Mitogen-activated protein kinases (MAPKs) are key mediators of the T cell receptor (TCR) signals but their roles in T helper (Th) cell differentiation are unclear. Here we showed that the MAPK kinase kinases MEKK2 (encoded by Map3k2) and MEKK3 (encoded by Map3k3) negatively regulated transforming growth factor-β (TGF-β)-mediated Th cell differentiation. Map3k2(-/-)Map3k3(Lck-Cre/-) mice showed an abnormal accumulation of regulatory T (Treg) and Th17 cells in the periphery, consistent with Map3k2(-/-)Map3k3(Lck-Cre/-) naive CD4(+) T cells' differentiation into Treg and Th17 cells with a higher frequency than wild-type (WT) cells after TGF-β stimulation in vitro. In addition, Map3k2(-/-)Map3k3(Lck-Cre/-) mice developed more severe experimental autoimmune encephalomyelitis. Map3k2(-/-)Map3k3(Lck-Cre/-) T cells exhibited impaired phosphorylation of SMAD2 and SMAD3 proteins at their linker regions, which negatively regulated the TGF-β responses in T cells. Thus, the crosstalk between TCR-induced MAPK and the TGF-β signaling pathways is important in regulating Th cell differentiation. Copyright © 2011 Elsevier Inc. All rights reserved.

Administration of herpes simplex-thymidine kinase-expressing donor T cells with a T-cell-depleted allogeneic marrow graft.

PubMed

Tiberghien, P; Ferrand, C; Lioure, B; Milpied, N; Angonin, R; Deconinck, E; Certoux, J M; Robinet, E; Saas, P; Petracca, B; Juttner, C; Reynolds, C W; Longo, D L; Hervé, P; Cahn, J Y

2001-01-01

Administration of donor T cells expressing the herpes simplex-thymidine kinase (HS-tk) with a hematopoietic stem cell (HSC) transplantation could allow, if graft-versus-host disease (GVHD) was to occur, a selective in vivo depletion of these T cells by the use of ganciclovir (GCV). The study evaluates the feasibility of such an approach. Escalating numbers of donor HS-tk-expressing CD3(+) gene-modified cells (GMCs) are infused with a T-cell-depleted bone marrow transplantation (BMT). Twelve patients with hematological malignancies received 2 x 10(5) (n = 5), 6 x 10(5) (n = 5), or 20 x 10(5) (n = 2) donor CD3(+) GMCs/kg with a BMT from a human leukocyte antigen (HLA)-identical sibling. No acute toxicity was associated with GMC administration. An early increase of circulating GMCs followed by a progressive decrease and long-lasting circulation of GMCs was documented. GCV treatment resulted in significant rapid decrease in circulating GMCs. Three patients developed acute GVHD, with a grade of at least II, while one patient developed chronic GVHD. Treatment with GCV alone was associated with a complete remission (CR) in 2 patients with acute GVHD, while the addition of glucocorticoids was necessary to achieve a CR in the last case. Long-lasting CR occurred with GCV treatment in the patient with chronic GVHD. Unfortunately, Epstein-Barr virus-lymphoproliferative disease occurred in 3 patients. Overall, the administration of low numbers of HS-tk-expressing T cells early following an HLA-identical BMT is associated with no acute toxicity, persistent circulation of the GMCs, and GCV-sensitive GVHD. Such findings open the way to the infusion of higher numbers of gene-modified donor T cells to enhance post-BMT immune competence while preserving GCV-sensitive alloreactivity.

The C-Type Lectin OCILRP2 Costimulates EL4 T Cell Activation via the DAP12-Raf-MAP Kinase Pathway

PubMed Central

Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

2014-01-01

OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation. PMID:25411776

The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck.

PubMed Central

Raab, M; Yamamoto, M; Rudd, C E

1994-01-01

CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth. CD5 associates with the T-cell receptor zeta chain (TcR zeta)/CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta/CD3 ligation. However, despite this, the mechanism by which CD5 generates intracellular signals is unclear. In this study, we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck. Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues. Further, anti-CD5 and anti-p56lck coprecipitated each other in a variety of detergents, including Nonidet P-40 and Triton X-100. Anti-CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta/CD3 or CD4. No binding between p59fyn(T) and CD5 was detected in T cells. The binding of p56lck to CD5 induced a 10- to 15-fold increase in p56lck catalytic activity, as measured by in vitro kinase analysis. In vivo labelling with 32P(i) also showed a four- to fivefold increase in Y-394 occupancy in p56lck when associated with CD5. The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system. CD5 interaction with p56lck represents a novel variant of a receptor-kinase complex in which receptor can also serve as substrate. The CD5-p56lck interaction is likely to play roles in T-cell signalling and T-B collaboration. Images PMID:7513045

Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8+ Cytolytic T Cell Responses

PubMed Central

Taylor, Alison; Harker, James A.; Chanthong, Kittiphat; Stevenson, Philip G.; Zuniga, Elina I.; Rudd, Christopher E.

2016-01-01

Summary Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and β) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8+ T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8+ cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8+ CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8+ OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy. PMID:26885856

LKB1 tumor suppressor and salt-inducible kinases negatively regulate human T-cell leukemia virus type 1 transcription

PubMed Central

2013-01-01

Background Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Treatment options are limited and prophylactic agents are not available. We have previously demonstrated an essential role for CREB-regulating transcriptional coactivators (CRTCs) in HTLV-1 transcription. Results In this study we report on the negative regulatory role of LKB1 tumor suppressor and salt-inducible kinases (SIKs) in the activation of HTLV-1 long terminal repeats (LTR) by the oncoprotein Tax. Activation of LKB1 and SIKs effectively blunted Tax activity in a phosphorylation-dependent manner, whereas compromising these kinases, but not AMP-dependent protein kinases, augmented Tax function. Activated LKB1 and SIKs associated with Tax and suppressed Tax-induced LTR activation by counteracting CRTCs and CREB. Enforced expression of LKB1 or SIK1 in cells transfected with HTLV-1 molecular clone pX1MT repressed proviral transcription. On the contrary, depletion of LKB1 in pX1MT-transfected cells and in HTLV-1-transformed T cells boosted the expression of Tax. Treatment of HTLV-1 transformed cells with metformin led to LKB1/SIK1 activation, reduction in Tax expression, and inhibition of cell proliferation. Conclusions Our findings revealed a new function of LKB1 and SIKs as negative regulators of HTLV-1 transcription. Pharmaceutical activation of LKB1 and SIKs might be considered as a new strategy in anti-HTLV-1 and anti-ATL therapy. PMID:23577667

Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.

PubMed

Bubeck Wardenburg, J; Fu, C; Jackman, J K; Flotow, H; Wilkinson, S E; Williams, D H; Johnson, R; Kong, G; Chan, A C; Findell, P R

1996-08-16

Two families of tyrosine kinases, the Src and Syk families, are required for T-cell receptor activation. While the Src kinases are responsible for phosphorylation of receptor-encoded signaling motifs and for up-regulation of ZAP-70 activity, the downstream substrates of ZAP-70 are unknown. Evidence is presented herein that the Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is a substrate of ZAP-70. Phosphorylation of SLP-76 is diminished in T cells that express a catalytically inactive ZAP-70. Moreover, SLP-76 is preferentially phosphorylated by ZAP-70 in vitro and in heterologous cellular systems. In T cells, overexpression of wild-type SLP-76 results in a hyperactive receptor, while expression of a SLP-76 molecule that is unable to be tyrosine-phosphorylated attenuates receptor function. In addition, the SH2 domain of SLP-76 is required for T-cell receptor function, although its role is independent of the ability of SLP-76 to undergo tyrosine phosphorylation. As SLP-76 interacts with both Grb2 and phospholipase C-gamma1, these data indicate that phosphorylation of SLP-76 by ZAP-70 provides an important functional link between the T-cell receptor and activation of ras and calcium pathways.

Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo.

PubMed

Ulges, Alexander; Klein, Matthias; Reuter, Sebastian; Gerlitzki, Bastian; Hoffmann, Markus; Grebe, Nadine; Staudt, Valérie; Stergiou, Natascha; Bohn, Toszka; Brühl, Till-Julius; Muth, Sabine; Yurugi, Hajime; Rajalingam, Krishnaraj; Bellinghausen, Iris; Tuettenberg, Andrea; Hahn, Susanne; Reißig, Sonja; Haben, Irma; Zipp, Frauke; Waisman, Ari; Probst, Hans-Christian; Beilhack, Andreas; Buchou, Thierry; Filhol-Cochet, Odile; Boldyreff, Brigitte; Breloer, Minka; Jonuleit, Helmut; Schild, Hansjörg; Schmitt, Edgar; Bopp, Tobias

2015-03-01

The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the β-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.

A new strips tracker for the upgraded ATLAS ITk detector

NASA Astrophysics Data System (ADS)

David, C.

2018-01-01

The ATLAS detector has been designed and developed to function in the environment of the present Large Hadron Collider (LHC). At the next-generation tracking detector proposed for the High Luminosity LHC (HL-LHC), the so-called ATLAS Phase-II Upgrade, the fluences and radiation levels will be higher by as much as a factor of ten. The new sub-detectors must thus be faster, of larger area, more segmented and more radiation hard while the amount of inactive material should be minimized and the power supply to the front-end systems should be increased. For those reasons, the current inner tracker of the ATLAS detector will be fully replaced by an all-silicon tracking system that consists of a pixel detector at small radius close to the beam line and a large area strip tracker surrounding it. This document gives an overview of the design of the strip inner tracker (Strip ITk) and summarises the intensive R&D activities performed over the last years by the numerous institutes within the Strips ITk collaboration. These studies are accompanied with a strong prototyping effort to contribute to the optimisation of the Strip ITk's structure and components. This effort culminated recently in the release of the ATLAS Strips ITk Technical Design Report (TDR).

Dysregulated choline metabolism in T-cell lymphoma: role of choline kinase-α and therapeutic targeting

PubMed Central

Xiong, J; Bian, J; Wang, L; Zhou, J-Y; Wang, Y; Zhao, Y; Wu, L-L; Hu, J-J; Li, B; Chen, S-J; Yan, C; Zhao, W-L

2015-01-01

Cancer cells have distinct metabolomic profile. Metabolic enzymes regulate key oncogenic signaling pathways and have an essential role on tumor progression. Here, serum metabolomic analysis was performed in 45 patients with T-cell lymphoma (TCL) and 50 healthy volunteers. The results showed that dysregulation of choline metabolism occurred in TCL and was related to tumor cell overexpression of choline kinase-α (Chokα). In T-lymphoma cells, pharmacological and molecular silencing of Chokα significantly decreased Ras-GTP activity, AKT and ERK phosphorylation and MYC oncoprotein expression, leading to restoration of choline metabolites and induction of tumor cell apoptosis/necropotosis. In a T-lymphoma xenograft murine model, Chokα inhibitor CK37 remarkably retarded tumor growth, suppressed Ras-AKT/ERK signaling, increased lysophosphatidylcholine levels and induced in situ cell apoptosis/necropotosis. Collectively, as a regulatory gene of aberrant choline metabolism, Chokα possessed oncogenic activity and could be a potential therapeutic target in TCL, as well as other hematological malignancies with interrupted Ras signaling pathways. PMID:25768400

Module and electronics developments for the ATLAS ITk pixel system

NASA Astrophysics Data System (ADS)

Muñoz, F. J.

2018-03-01

The ATLAS experiment is preparing for an extensive modification of its detectors in the course of the planned HL-LHC accelerator upgrade around 2025. The ATLAS upgrade includes the replacement of the entire tracking system by an all-silicon detector (Inner Tracker, ITk). The five innermost layers of ITk will be a pixel detector built of new sensor and readout electronics technologies to improve the tracking performance and cope with the severe HL-LHC environment in terms of occupancy and radiation. The total area of the new pixel system could measure up to 14 m2, depending on the final layout choice, which is expected to take place in 2018. In this paper an overview of the ongoing R&D activities on modules and electronics for the ATLAS ITk is given including the main developments and achievements in silicon planar and 3D sensor technologies, readout and power challenges.

Glycycoumarin exerts anti-liver cancer activity by directly targeting T-LAK cell-originated protein kinase.

PubMed

Song, Xinhua; Yin, Shutao; Zhang, Enxiang; Fan, Lihong; Ye, Min; Zhang, Yong; Hu, Hongbo

2016-10-04

Glycycoumarin (GCM) is a major bioactive coumarin compound isolated from licorice and the anti-cancer activity of GCM has not been scientifically addressed. In the present study, we have tested the anti-liver cancer activity of GCM using both in vitro and in vivo models and found for the first time that GCM possesses a potent activity against liver cancer evidenced by cell growth inhibition and apoptosis induction in vitro and tumor reduction in vivo. Mechanistically, GCM was able to bind to and inactivate oncogenic kinase T-LAK cell-originated protein kinase (TOPK), which in turn led to activation of p53 pathway. Our findings supported GCM as a novel active compound that contributed to the anti-cancer activity of licorice and TOPK could be an effective target for hepatocellular carcinoma (HCC) treatment.

Ibrutinib treatment ameliorates murine chronic graft-versus-host disease

PubMed Central

Dubovsky, Jason A.; Flynn, Ryan; Du, Jing; Harrington, Bonnie K.; Zhong, Yiming; Kaffenberger, Benjamin; Yang, Carrie; Towns, William H.; Lehman, Amy; Johnson, Amy J.; Muthusamy, Natarajan; Devine, Steven M.; Jaglowski, Samantha; Serody, Jonathan S.; Murphy, William J.; Munn, David H.; Luznik, Leo; Hill, Geoffrey R.; Wong, Henry K.; MacDonald, Kelli K.P.; Maillard, Ivan; Koreth, John; Elias, Laurence; Cutler, Corey; Soiffer, Robert J.; Antin, Joseph H.; Ritz, Jerome; Panoskaltsis-Mortari, Angela; Byrd, John C.; Blazar, Bruce R.

2014-01-01

Chronic graft-versus-host disease (cGVHD) is a life-threatening impediment to allogeneic hematopoietic stem cell transplantation, and current therapies do not completely prevent and/or treat cGVHD. CD4+ T cells and B cells mediate cGVHD; therefore, targeting these populations may inhibit cGVHD pathogenesis. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton’s tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) that targets Th2 cells and B cells and produces durable remissions in B cell malignancies with minimal toxicity. Here, we evaluated whether ibrutinib could reverse established cGVHD in 2 complementary murine models, a model interrogating T cell–driven sclerodermatous cGVHD and an alloantibody-driven multiorgan system cGVHD model that induces bronchiolar obliterans (BO). In the T cell–mediated sclerodermatous cGVHD model, ibrutinib treatment delayed progression, improved survival, and ameliorated clinical and pathological manifestations. In the alloantibody-driven cGVHD model, ibrutinib treatment restored pulmonary function and reduced germinal center reactions and tissue immunoglobulin deposition. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Furthermore, ibrutinib treatment reduced activation of T and B cells from patients with active cGVHD. Our data demonstrate that B cells and T cells drive cGVHD and suggest that ibrutinib has potential as a therapeutic agent, warranting consideration for cGVHD clinical trials. PMID:25271622

SPAK kinase is a substrate and target of PKCθ in T-cell receptor-induced AP-1 activation pathway

PubMed Central

Li, Yingqiu; Hu, Junru; Vita, Randi; Sun, Binggang; Tabata, Hiroki; Altman, Amnon

2004-01-01

Protein kinase C-θ (PKCθ) plays an important role in T-cell activation via stimulation of AP-1 and NF-κB. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKCθ-interacting kinase. SPAK interacted with PKCθ (but not with PKCα) via its 99 COOH-terminal residues. TCR/CD28 costimulation enhanced this association and stimulated the catalytic activity of SPAK. Recombinant SPAK was phosphorylated on Ser-311 in its kinase domain by PKCθ, but not by PKCα. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCθ-deficient T cells. Transfected SPAK synergized with constitutively active PKCθ to activate AP-1, but not NF-κB. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKCθ-interacting region of SPAK, and Ser-311 mutation greatly reduced these activities of SPAK. Conversely, a SPAK-specific RNAi or a dominant-negative SPAK mutant inhibited PKCθ- and TCR/CD28-induced AP-1, but not NF-κB, activation. These results define SPAK as a substrate and target of PKCθ in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-κB) activation. PMID:14988727

Signaling network of the Btk family kinases.

PubMed

Qiu, Y; Kung, H J

2000-11-20

The Btk family kinases represent new members of non-receptor tyrosine kinases, which include Btk/Atk, Itk/Emt/Tsk, Bmx/Etk, and Tec. They are characterized by having four structural modules: PH (pleckstrin homology) domain, SH3 (Src homology 3) domain, SH2 (Src homology 2) domain and kinase (Src homology 1) domain. Increasing evidence suggests that, like Src-family kinases, Btk family kinases play central but diverse modulatory roles in various cellular processes. They participate in signal transduction in response to virtually all types of extracellular stimuli which are transmitted by growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen-receptors and integrins. They are regulated by many non-receptor tyrosine kinases such as Src, Jak, Syk and FAK family kinases. In turn, they regulate many of major signaling pathways including those of PI3K, PLCgamma and PKC. Both genetic and biochemical approaches have been used to dissect the signaling pathways and elucidate their roles in growth, differentiation and apoptosis. An emerging new role of this family of kinases is cytoskeletal reorganization and cell motility. The physiological importance of these kinases was amply demonstrated by their link to the development of immunodeficiency diseases, due to germ-line mutations. The present article attempts to review the structure and functions of Btk family kinases by summarizing our current knowledge on the interacting partners associated with the different modules of the kinases and the diverse signaling pathways in which they are involved.

Therapeutic antitumor immunity by checkpoint blockade is enhanced by ibrutinib, an inhibitor of both BTK and ITK.

PubMed

Sagiv-Barfi, Idit; Kohrt, Holbrook E K; Czerwinski, Debra K; Ng, Patrick P; Chang, Betty Y; Levy, Ronald

2015-03-03

Monoclonal antibodies can block cellular interactions that negatively regulate T-cell immune responses, such as CD80/CTLA-4 and PD-1/PD1-L, amplifying preexisting immunity and thereby evoking antitumor immune responses. Ibrutinib, an approved therapy for B-cell malignancies, is a covalent inhibitor of BTK, a member of the B-cell receptor (BCR) signaling pathway, which is critical to the survival of malignant B cells. Interestingly this drug also inhibits ITK, an essential enzyme in Th2 T cells and by doing so it can shift the balance between Th1 and Th2 T cells and potentially enhance antitumor immune responses. Here we report that the combination of anti-PD-L1 antibody and ibrutinib suppresses tumor growth in mouse models of lymphoma that are intrinsically insensitive to ibrutinib. The combined effect of these two agents was also documented for models of solid tumors, such as triple negative breast cancer and colon cancer. The enhanced therapeutic activity of PD-L1 blockade by ibrutinib was accompanied by enhanced antitumor T-cell immune responses. These preclinical results suggest that the combination of PD1/PD1-L blockade and ibrutinib should be tested in the clinic for the therapy not only of lymphoma but also in other hematologic malignancies and solid tumors that do not even express BTK.

Dual role of SLP-76 in mediating T cell receptor-induced activation of phospholipase C-gamma1.

PubMed

Beach, Dvora; Gonen, Ronnie; Bogin, Yaron; Reischl, Ilona G; Yablonski, Deborah

2007-02-02

Phospholipase C-gamma1 (PLC-gamma1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-gamma1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-gamma1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-gamma1 GEM recruitment, but were required for PLC-gamma1 phosphorylation at Tyr(783). Thus, GEM recruitment can be insufficient for full activation of PLC-gamma1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-gamma1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-gamma1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-gamma1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.

CD6 and Linker of Activated T Cells are Potential Interaction Partners for T Cell-Specific Adaptor Protein.

PubMed

Hem, C D; Ekornhol, M; Granum, S; Sundvold-Gjerstad, V; Spurkland, A

2017-02-01

The T cell-specific adaptor protein (TSAd) contains several protein interaction domains, and is merging as a modulator of T cell activation. Several interaction partners for the TSAd proline-rich region and phosphotyrosines have been identified, including the Src and Tec family kinases lymphocyte-specific protein tyrosine kinase and interleukin 2-inducible T cell kinase. Via its Src homology 2 (SH2) domain, TSAd may thus function as a link between these enzymes and other signalling molecules. However, few binding partners to the TSAd SH2 domain in T cells are hitherto known. Through the use of in silico ligand prediction, peptide spot arrays, pull-down and immunoprecipitation experiments, we here report novel interactions between the TSAd SH2 domain and CD6 phosphotyrosine (pTyr) 629 and linker of activated T cells (LAT) pTyr 171 , pTyr 191 and pTyr 226 . © 2016 The Foundation for the Scandinavian Journal of Immunology.

Interleukins 2, 4, 7, and 15 stimulate tyrosine phosphorylation of insulin receptor substrates 1 and 2 in T cells. Potential role of JAK kinases.

PubMed

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.

An ITK framework for deterministic global optimization for medical image registration

NASA Astrophysics Data System (ADS)

Dru, Florence; Wachowiak, Mark P.; Peters, Terry M.

2006-03-01

Similarity metric optimization is an essential step in intensity-based rigid and nonrigid medical image registration. For clinical applications, such as image guidance of minimally invasive procedures, registration accuracy and efficiency are prime considerations. In addition, clinical utility is enhanced when registration is integrated into image analysis and visualization frameworks, such as the popular Insight Toolkit (ITK). ITK is an open source software environment increasingly used to aid the development, testing, and integration of new imaging algorithms. In this paper, we present a new ITK-based implementation of the DIRECT (Dividing Rectangles) deterministic global optimization algorithm for medical image registration. Previously, it has been shown that DIRECT improves the capture range and accuracy for rigid registration. Our ITK class also contains enhancements over the original DIRECT algorithm by improving stopping criteria, adaptively adjusting a locality parameter, and by incorporating Powell's method for local refinement. 3D-3D registration experiments with ground-truth brain volumes and clinical cardiac volumes show that combining DIRECT with Powell's method improves registration accuracy over Powell's method used alone, is less sensitive to initial misorientation errors, and, with the new stopping criteria, facilitates adequate exploration of the search space without expending expensive iterations on non-improving function evaluations. Finally, in this framework, a new parallel implementation for computing mutual information is presented, resulting in near-linear speedup with two processors.

3'-Azido-2',3'-dideoxythymidine induced deficiency of thymidine kinases 1, 2 and deoxycytidine kinase in H9 T-lymphoid cells.

PubMed

Gröschel, Bettina; Kaufmann, Andreas; Höver, Gerold; Cinatl, Jaroslav; Doerr, Hans Wilhelm; Noordhuis, Paul; Loves, Willem J P; Peters, Godefridus J; Cinatl, Jindrich

2002-07-15

Continuous cultivation of T-lymphoid H9 cells in the presence of 3'-azido-2',3'-dideoxythymidine (AZT) resulted in a cell variant cross-resistant to both thymidine and deoxycytidine analogs. Cytotoxic effects of AZT, 2',3'-didehydro-3'-deoxythymidine as well as different deoxycytidine analogs such as 2',3'-dideoxycytidine, 2',2'-difluoro-2'-deoxycytidine (dFdC) and 1-ss-D-arabinofuranosylcytosine (Ara-C) were strongly reduced in H9 cells continuously exposed to AZT when compared to parental cells (>8.3-, >6.6-, >9.1-, 5 x 10(4)-, 5 x 10(3)-fold, respectively). Moreover, anti-HIV-1 effects of AZT, d4T, ddC and 2',3'-dideoxy-3'-thiacytidine (3TC) were significantly diminished (>222-, >25-, >400-, >200-fold, respectively) in AZT-resistant H9 cells. Study of cellular mechanisms responsible for cross-resistance to pyrimidine analogs in AZT-resistant H9 cells revealed decreased mRNA levels of thymidine kinase 1 (TK1) and lack of deoxycytidine kinase (dCK) mRNA expression. The loss of dCK gene expression was confirmed by western blot analysis of dCK protein as well as dCK enzyme activity assay. Moreover, enzyme activity of TK1 and TK2 was reduced in AZT-resistant cells. In order to determine whether lack of dCK affected the formation of the active triphosphate of the deoxycytidine analog dFdC, dFdCTP accumulation and retention was measured in H9 parental and AZT-resistant cells after exposure to 1 and 10 microM dFdC. Parental H9 cells accumulated about 30 and 100 pmol dFdCTP/10(6) cells after 4hr, whereas in AZT-resistant cells no dFdCTP accumulation was detected. These results demonstrate that continuous treatment of H9 cells in the presence of AZT selected for a thymidine analog resistant cell variant with cross-resistance to deoxycytidine analogs, due to deficiency in TK1, TK2, and dCK.

Phosphorylation of the Grb2- and phosphatidylinositol 3-kinase p85-binding p36/38 by Syk in Lck-negative T cells.

PubMed

von Willebrand, M; Williams, S; Tailor, P; Mustelin, T

1998-06-01

Activation of the mitogen-activated protein kinase (MAPK) pathway by the T-cell antigen receptor (TCR) in T cells involves a positive role for phosphatidylinositol 3-kinase (PI3K) activity. We recently reported that over-expression of the Syk protein tyrosine kinase in the Lck-negative JCaM1 cells enabled the TCR to induce a normal activation of the Erk2 MAPK and enhanced transcription of a reporter gene driven by the nuclear factor of activated T cells and AP-1. Because this system allows us to analyse the targets for Syk in receptor-mediated signalling, we examined the role of PI3K in signalling events between the TCR-regulated Syk and the downstream activation of Erk2. We report that inhibition of PI3K by wortmannin or an inhibitory p85 construct, p85deltaiSH2, reduced the TCR-induced Syk-dependent activation of Erk2, as well as the appearance of phospho-Erk and phospho-Mek. At the same time, expression of Syk resulted in the activation-dependent phosphorylation of three proteins that bound to the src homology 2 (SH2) domains of PI3K p85. The strongest of these bands had an apparent molecular mass of 36-38 kDa on SDS gels, and it was quantitatively removed from the lysates by adsorption to a fusion protein containing the SH2 domain of Grb2. The appearance of this band was Syk dependent, and it was seen only upon triggering of the TCR complex. Thus, p36/38 was phosphorylated by Syk or a Syk-regulated kinase, and this protein may provide a link to the recruitment and activation of PI3K, as well as to the Ras-MAPK pathway, in TCR-triggered T cells.

T-LAK cell-originated protein kinase presents a novel therapeutic target in FLT3-ITD mutated acute myeloid leukemia.

PubMed

Alachkar, Houda; Mutonga, Martin; Malnassy, Gregory; Park, Jae-Hyun; Fulton, Noreen; Woods, Alex; Meng, Liping; Kline, Justin; Raca, Gordana; Odenike, Olatoyosi; Takamatsu, Naofumi; Miyamoto, Takashi; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2015-10-20

Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML.

T-LAK cell-originated protein kinase presents a novel therapeutic target in FLT3-ITD mutated acute myeloid leukemia

PubMed Central

Alachkar, Houda; Mutonga, Martin; Malnassy, Gregory; Park, Jae-Hyun; Fulton, Noreen; Woods, Alex; Meng, Liping; Kline, Justin; Raca, Gordana; Odenike, Olatoyosi; Takamatsu, Naofumi; Miyamoto, Takashi; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2015-01-01

Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML. PMID:26450903

The T-LAK Cell-originated Protein Kinase Signal Pathway Promotes Colorectal Cancer Metastasis.

PubMed

Zykova, Tatyana A; Zhu, Feng; Wang, Lei; Li, Haitao; Bai, Ruihua; Lim, Do Young; Yao, Ke; Bode, Ann M; Dong, Zigang

2017-04-01

Approximately 90% of all cancer deaths arise from the metastatic dissemination of primary tumors. Metastasis is the most lethal attribute of colorectal cancer. New data regarding the molecules contributing to the metastatic phenotype, the pathways they control and the genes they regulate are very important for understanding the processes of metastasis prognosis and prevention in the clinic. The purpose of this study was to investigate the role of T-LAK cell-originated protein kinase (TOPK) in the promotion of colorectal cancer metastasis. TOPK is highly expressed in human metastatic colorectal cancer tissue compared with malignant adenocarcinoma. We identified p53-related protein kinase (PRPK) as a new substrate of TOPK. TOPK binds with and phosphorylates PRPK at Ser250 in vitro and ex vivo. This site plays a critical role in the function of PRPK. Cell lines stably expressing mutant PRPK (S250A), knockdown TOPK, knockdown PRPK or knockdown of both TOPK and PRPK significantly inhibited liver metastasis of human HCT116 colon cancer cells in a xenograft mouse model. Therefore, we conclude that TOPK directly promotes metastasis of colorectal cancer by modulating PRPK. Thus, these findings may assist in the prediction of prognosis or development of new therapeutic strategies against colon cancer. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Wenjun; Ercan, Dalia; Chen, Liang

2010-01-12

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potentmore » against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.« less

ITK: enabling reproducible research and open science

PubMed Central

McCormick, Matthew; Liu, Xiaoxiao; Jomier, Julien; Marion, Charles; Ibanez, Luis

2014-01-01

Reproducibility verification is essential to the practice of the scientific method. Researchers report their findings, which are strengthened as other independent groups in the scientific community share similar outcomes. In the many scientific fields where software has become a fundamental tool for capturing and analyzing data, this requirement of reproducibility implies that reliable and comprehensive software platforms and tools should be made available to the scientific community. The tools will empower them and the public to verify, through practice, the reproducibility of observations that are reported in the scientific literature. Medical image analysis is one of the fields in which the use of computational resources, both software and hardware, are an essential platform for performing experimental work. In this arena, the introduction of the Insight Toolkit (ITK) in 1999 has transformed the field and facilitates its progress by accelerating the rate at which algorithmic implementations are developed, tested, disseminated and improved. By building on the efficiency and quality of open source methodologies, ITK has provided the medical image community with an effective platform on which to build a daily workflow that incorporates the true scientific practices of reproducibility verification. This article describes the multiple tools, methodologies, and practices that the ITK community has adopted, refined, and followed during the past decade, in order to become one of the research communities with the most modern reproducibility verification infrastructure. For example, 207 contributors have created over 2400 unit tests that provide over 84% code line test coverage. The Insight Journal, an open publication journal associated with the toolkit, has seen over 360,000 publication downloads. The median normalized closeness centrality, a measure of knowledge flow, resulting from the distributed peer code review system was high, 0.46. PMID:24600387

ITK: enabling reproducible research and open science.

PubMed

McCormick, Matthew; Liu, Xiaoxiao; Jomier, Julien; Marion, Charles; Ibanez, Luis

2014-01-01

Reproducibility verification is essential to the practice of the scientific method. Researchers report their findings, which are strengthened as other independent groups in the scientific community share similar outcomes. In the many scientific fields where software has become a fundamental tool for capturing and analyzing data, this requirement of reproducibility implies that reliable and comprehensive software platforms and tools should be made available to the scientific community. The tools will empower them and the public to verify, through practice, the reproducibility of observations that are reported in the scientific literature. Medical image analysis is one of the fields in which the use of computational resources, both software and hardware, are an essential platform for performing experimental work. In this arena, the introduction of the Insight Toolkit (ITK) in 1999 has transformed the field and facilitates its progress by accelerating the rate at which algorithmic implementations are developed, tested, disseminated and improved. By building on the efficiency and quality of open source methodologies, ITK has provided the medical image community with an effective platform on which to build a daily workflow that incorporates the true scientific practices of reproducibility verification. This article describes the multiple tools, methodologies, and practices that the ITK community has adopted, refined, and followed during the past decade, in order to become one of the research communities with the most modern reproducibility verification infrastructure. For example, 207 contributors have created over 2400 unit tests that provide over 84% code line test coverage. The Insight Journal, an open publication journal associated with the toolkit, has seen over 360,000 publication downloads. The median normalized closeness centrality, a measure of knowledge flow, resulting from the distributed peer code review system was high, 0.46.

Tax-dependent stimulation of G1 phase-specific cyclin-dependent kinases and increased expression of signal transduction genes characterize HTLV type 1-transformed T cells.

PubMed

Haller, K; Ruckes, T; Schmitt, I; Saul, D; Derow, E; Grassmann, R

2000-11-01

Human T cell leukemia virus protein induces T cells to permanent IL-2-dependent growth. These cells occasionally convert to factor independence. The viral oncoprotein Tax acts as an essential growth factor of transformed lymphocytes and stimulates the cell cycle in the G(1) phase. In T cells and fibroblasts Tax enhances the activity of the cyclin-dependent kinases (CDK) CDK4 and CDK6. These kinases, which require binding to cyclin D isotypes for their activity, control the G(1) phase. Coimmunoprecipitation from these cells revealed that Tax associates with cyclin D3/CDK6, suggesting a direct activation of this kinase. The CDK stimulation may account in part for the mitogenic Tax effect, which causes IL-2-dependent T cell growth by Tax. To address the conversion to IL-2-independent proliferation and to identify overexpressed genes, which contribute to the transformed growth, the gene expression patterns of HTLV-1-transformed T cells were compared with that of peripheral blood lymphocytes. Potentially overexpressed cDNAs were cloned, sequenced, and used to determine the RNA expression. Genes found to be up-regulated are involved in signal transduction (STAT5a, cyclin G(1), c-fgr, hPGT) and also glycoprotein synthesis (LDLC, ribophorin). Many of these are also activated during T cell activation and implicated in the regulation of growth and apoptosis. The transcription factor STAT5a, which is involved in IL-2 signaling, was strongly up-regulated only in IL-2-independent cells, thus suggesting that it contributes to factor-independent growth. Thus, the differentially expressed genes could cooperate with the Tax-induced cell cycle stimulation in the maintenance of IL-2-dependent and IL-2-independent growth of HTLV-transformed lymphocytes.

Dysregulated miR34a/diacylglycerol kinase ζ interaction enhances T-cell activation in acquired aplastic anemia.

PubMed

Sun, Yuan-Xin; Li, Hui; Feng, Qi; Li, Xin; Yu, Ying-Yi; Zhou, Li-Wei; Gao, Yan; Li, Guo-Sheng; Ren, Juan; Ma, Chun-Hong; Gao, Cheng-Jiang; Peng, Jun

2017-01-24

Acquired aplastic anemia is an idiopathic paradigm of human bone marrow failure syndrome, which involves active destruction of hematopoietic stem cells and progenitors by cytotoxic T cells in the bone marrow. Aberrant expression of microRNAs in T cells has been shown to lead to development of certain autoimmune diseases. In the present study, we performed a microarray analysis of miRNA expression in bone marrow CD3+ T cells from patients with aplastic anemia and healthy controls. Overexpression of miR34a and underexpression of its target gene diacylglycerol kinase (DGK) ζ in bone marrow mononuclear cells were validated in 41 patients and associated with the severity of aplastic anemia. Further, the level of miR34a was higher in naïve T cells from patients than from controls. The role of miR34a and DGKζ in aplastic anemia was investigated in a murine model of immune-mediated bone marrow failure using miR34a-/- mice. After T-cell receptor stimulation in vitro, lymph node T cells from miR34a-/- mice demonstrated reduced activation and proliferation accompanied with a less profound down-regulation of DGKζ expression and decreased ERK phosphorylation compared to those from wild-type C57BL6 control mice. Infusion of 5 × 106 miR34a-/- lymph node T cells into sublethally irradiated CB6F1 recipients led to increased Lin-Sca1+CD117+ cells and less vigorous expansion of CD8+ T cells than injection of same number of wild-type lymph node cells. Our study demonstrates that the miR34a/DGKζ dysregulation enhances T-cell activation in aplastic anemia and targeting miR34a may represent a novel molecular therapeutic approach for patients with aplastic anemia.

Modulation of T-cell receptor functional sensitivity via the opposing actions of protein tyrosine kinases and phosphatases: a mathematical model.

PubMed

Szomolay, Barbara; van den Berg, Hugo A

2014-12-01

Combining receptor kinetics and stochastic modelling of receptor activation, we show that a T-cell can specifically augment its functional sensitivity to one particular peptide ligand while simultaneously decreasing its sensitivity to other ligands, by coordinating the expression levels of the co-receptor CD8 and the relative activities of kinases and phosphatases in the vicinity of the T-cell receptor (TCR). We propose that this focusable degeneracy of epitope recognition allows a TCR to have a wide range of potential ligands but be specifically sensitive to only one or a few of these at any one time, which resolves the paradox of how a relatively small number of clones (∼10(6)) can maintain the potential to respond to a vast space of ligands (∼20(9)) whilst avoiding auto-immunity. We validate the model against experimental data and predict shifts in functional sensitivity following a shift in the kinase/phosphatase balance (which could in principle be induced by experimental means). Moreover, we propose that in vivo, the T-cell gauges ligand quality by monitoring changes in TCR triggering rate concomitant with shifts in this balance, for instance as the immunological synapse matures.

Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

NASA Technical Reports Server (NTRS)

Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

2002-01-01

Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

Turning behaviors of T cells climbing up ramp-like structures are regulated by myosin light chain kinase activity and lamellipodia formation.

PubMed

Song, Kwang Hoon; Lee, Jaehyun; Jung, Hong-Ryul; Park, HyoungJun; Doh, Junsang

2017-09-14

T cells navigate diverse microenvironments to perform immune responses. Micro-scale topographical structures within the tissues, which may inherently exist in normal tissues or may be formed by inflammation or injury, can influence T cell migration, but how T cell migration is affected by such topographical structures have not been investigated. In this study, we fabricated ramp-like structures with a 5 μm height and various slopes, and observed T cells climbing up the ramp-like structures. T cells encountering the ramp-like structures exhibited MLC accumulation near head-tail junctions contacting the ramp-like structures, and made turns to the direction perpendicular to the ramp-like structures. Pharmacological study revealed that lamellipodia formation mediated by arp2/3 and contractility regulated by myosin light chain kinase (MLCK) were responsible for the intriguing turning behavior of T cells climbing the ramp-like structures. Arp2/3 or MLCK inhibition substantially reduced probability of T cells climbing sharp-edged ramp-like structures, indicating intriguing turning behavior of T cells mediated by lamellipodia formation and MLCK activity may be important for T cells to access inflamed or injured tissues with abrupt topographical changes.

Cefradine blocks solar-ultraviolet induced skin inflammation through direct inhibition of T-LAK cell-originated protein kinase

PubMed Central

Ke, Changshu; Zhang, Guiping; Xiao, Juanjuan; Wu, Dan; Zeng, Xiaoyu; Chen, Jingwen; Guo, Jinguang; Zhou, Jie; Shi, Fei; Zhu, Feng

2016-01-01

Skin inflammation, and skin cancer induced by excessive solar ultraviolet (SUV) is a great threat to human health. SUV induced skin inflammation through activating p38 mitogen-activated protein kinase (p38) and c-Jun N-termeinal kinases (JNKs). T-LAK cell-originated protein kinase (TOPK) plays an important role in this process. Herein, the clinical data showed TOPK, phospho-p38, phospho-JNKs were highly expressed in human solar dermatitis. Ex vivo studies showed that SUV induced the phosphorylation of p38 and JNKs in HaCat and JB6 cells in a dose and time dependent manner. Molecule docking model indicated cefradine, an FDA-approved cephalosporin antibiotic, directly binds with TOPK. The result of in vitro binding assay verified cefradine can directly bind with TOPK. In vitro kinase results showed cefradine can inhibit TOPK activity. Ex vivo studies further showed cefradine inhibited SUV-induced the phosphorylation level of p38, JNKs and H2AX through inhibiting TOPK activity in a dose and time dependent manner, and cefradine inhibited the secretion of IL6 and TNF-α in HaCat and JB6 cells. In vivo studies showed that cefradine down-regulated SUV-induced the phosphorylation of p38, JNKs and H2AX and inhibited the secretion of IL6 and TNF-α in Babl/c mice. These results indicated that cefradine can inhibit SUV-induced skin inflammation by blocking TOPK signaling pathway, and TOPK is an effective target for suppressing inflammation induced by SUV irradiation. PMID:27016423

SimpleITK Image-Analysis Notebooks: a Collaborative Environment for Education and Reproducible Research.

PubMed

Yaniv, Ziv; Lowekamp, Bradley C; Johnson, Hans J; Beare, Richard

2018-06-01

Modern scientific endeavors increasingly require team collaborations to construct and interpret complex computational workflows. This work describes an image-analysis environment that supports the use of computational tools that facilitate reproducible research and support scientists with varying levels of software development skills. The Jupyter notebook web application is the basis of an environment that enables flexible, well-documented, and reproducible workflows via literate programming. Image-analysis software development is made accessible to scientists with varying levels of programming experience via the use of the SimpleITK toolkit, a simplified interface to the Insight Segmentation and Registration Toolkit. Additional features of the development environment include user friendly data sharing using online data repositories and a testing framework that facilitates code maintenance. SimpleITK provides a large number of examples illustrating educational and research-oriented image analysis workflows for free download from GitHub under an Apache 2.0 license: github.com/InsightSoftwareConsortium/SimpleITK-Notebooks .

Protein Kinase C δ (PKCδ) Splice Variants Modulate Apoptosis Pathway in 3T3L1 Cells during Adipogenesis

PubMed Central

Patel, Rekha; Apostolatos, André; Carter, Gay; Ajmo, Joanne; Gali, Meghanath; Cooper, Denise R.; You, Min; Bisht, Kirpal S.; Patel, Niketa A.

2013-01-01

Increased food intake and lack of physical activity results in excess energy stored in adipocytes, and this imbalance contributes to obesity. New adipocytes are required for storage of energy in the white adipose tissue. This process of adipogenesis is widely studied in differentiating 3T3L1 preadipocytes in vitro. We have identified a key signaling kinase, protein kinase C delta (PKCδ), whose alternative splice variant expression is modulated during adipogenesis. We demonstrate that PKCδII splice variant promotes survival in differentiating 3T3L1 cells through the Bcl2 pathway. Here we demonstrate that resveratrol, a naturally occurring polyphenol, increases apoptosis and inhibits adipogenesis along with disruption of PKCδ alternative splicing during 3T3L1 differentiation. Importantly, we have identified a PKCδII splice variant inhibitor. This inhibitor may be a valuable tool with therapeutic implications in obesity. PMID:23902767

Proton pump inhibitor ilaprazole suppresses cancer growth by targeting T-cell-originated protein kinase

PubMed Central

Gao, Suyu; Cheng, Li; Hao, Bin; Li, Jiacheng; Chen, Yao; Hou, Xuemei; Chen, Lixia; Li, Hua

2017-01-01

T-cell-originated protein kinase (TOPK) is highly and frequently expressed in various cancer tissues and plays an indispensable role in the mitosis of cancer cells, and therefore, it is an important target for drug treatment of tumor. Ilaprazole was identified to be a potent TOPK inhibitor. The data indicated that ilaprazole inhibited TOPK activities with high affinity and selectivity. In vitro studies showed that ilaprazole inhibited TOPK activities in HCT116, ES-2, A549, SW1990 cancer cells. Moreover, knockdown of TOPK in these cells decreased their sensitivities to ilaprazole. Results of an in vivo study demonstrated that gavage of ilaprazole in HCT116 colon tumor-bearing mice effectively suppressed cancer growth. The TOPK downstream signaling molecule phospho-histone H3 in tumor tissues was also decreased after ilaprazole treatment. Our results suggested that ilaprazole inhibited the cancer growth by targeting TOPK both in vitro and in vivo. PMID:28388576

Physical interaction of human T-cell leukemia virus type 1 Tax with cyclin-dependent kinase 4 stimulates the phosphorylation of retinoblastoma protein.

PubMed

Haller, Kerstin; Wu, Yalin; Derow, Elisabeth; Schmitt, Iris; Jeang, Kuan-Teh; Grassmann, Ralph

2002-05-01

The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G(1) phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21(CIP). Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein.

ATM kinase inhibition in glial cells activates the innate immune response and causes neurodegeneration in Drosophila.

PubMed

Petersen, Andrew J; Rimkus, Stacey A; Wassarman, David A

2012-03-13

To investigate the mechanistic basis for central nervous system (CNS) neurodegeneration in the disease ataxia-telangiectasia (A-T), we analyzed flies mutant for the causative gene A-T mutated (ATM). ATM encodes a protein kinase that functions to monitor the genomic integrity of cells and control cell cycle, DNA repair, and apoptosis programs. Mutation of the C-terminal amino acid in Drosophila ATM inhibited the kinase activity and caused neuron and glial cell death in the adult brain and a reduction in mobility and longevity. These data indicate that reduced ATM kinase activity is sufficient to cause neurodegeneration in A-T. ATM kinase mutant flies also had elevated expression of innate immune response genes in glial cells. ATM knockdown in glial cells, but not neurons, was sufficient to cause neuron and glial cell death, a reduction in mobility and longevity, and elevated expression of innate immune response genes in glial cells, indicating that a non-cell-autonomous mechanism contributes to neurodegeneration in A-T. Taken together, these data suggest that early-onset CNS neurodegeneration in A-T is similar to late-onset CNS neurodegeneration in diseases such as Alzheimer's in which uncontrolled inflammatory response mediated by glial cells drives neurodegeneration.

The Phase-II ATLAS ITk pixel upgrade

NASA Astrophysics Data System (ADS)

Terzo, S.

2017-07-01

The entire tracking system of the ATLAS experiment will be replaced during the LHC Phase-II shutdown (foreseen to take place around 2025) by an all-silicon detector called the ``ITk'' (Inner Tracker). The innermost portion of ITk will consist of a pixel detector with five layers in the barrel region and ring-shaped supports in the end-cap regions. It will be instrumented with new sensor and readout electronics technologies to improve the tracking performance and cope with the HL-LHC environment, which will be severe in terms of occupancy and radiation levels. The new pixel system could include up to 14 m2 of silicon, depending on the final layout, which is expected to be decided in 2017. Several layout options are being investigated at the moment, including some with novel inclined support structures in the barrel end-cap overlap region and others with very long innermost barrel layers. Forward coverage could be as high as |eta| <4. Supporting structures will be based on low mass, highly stable and highly thermally conductive carbon-based materials cooled by evaporative carbon dioxide circulated in thin-walled titanium pipes embedded in the structures. Planar, 3D, and CMOS sensors are being investigated to identify the optimal technology, which may be different for the various layers. The RD53 Collaboration is developing the new readout chip. The pixel off-detector readout electronics will be implemented in the framework of the general ATLAS trigger and DAQ system. A readout speed of up to 5 Gb/s per data link will be needed in the innermost layers going down to 640 Mb/s for the outermost. Because of the very high radiation level inside the detector, the first part of the transmission has to be implemented electrically, with signals converted for optical transmission at larger radii. Extensive tests are being carried out to prove the feasibility of implementing serial powering, which has been chosen as the baseline for the ITk pixel system due to the reduced

Effects of overexpression of IL-1 receptor-associated kinase on NFkappaB activation, IL-2 production and stress-activated protein kinases in the murine T cell line EL4.

PubMed

Knop, J; Wesche, H; Lang, D; Martin, M U

1998-10-01

The association and activation of the IL-1 receptor-associated protein kinase (IRAK) to the IL-1 receptor complex is one of the earliest events detectable in IL-1 signal transduction. We generated permanent clones of the murine T cell line EL4 6.1 overexpressing human (h)IRAK to evaluate the role of this kinase in IL-1 signaling. Overexpression of hIRAK enhanced IL-1-stimulated activation of the transcription factor NFkappaB, whereas a truncated form (N-IRAK) specifically inhibited IL-1-dependent NFkappaB activity. In clones stably overexpressing hIRAK a weak constitutive activation of NFkappaB correlated with a low basal IL-2 production which was enhanced in an IL-1-dependent manner. Compared to the parental cell line the dose-response curve of IL-1-induced IL-2 production was shifted in both potency and efficacy. These results demonstrate that IRAK directly triggers NFkappaB-mediated gene expression in EL4 cells. Qualitatively different effects were observed for the IL-1-induced activation of stress-activated protein (SAP) kinases: permanent overexpression of IRAK did not affect the dose dependence but prolonged the kinetics of IL-1-induced activation of SAP kinases, suggesting that this signaling branch may be regulated by distinct mechanisms.

Regulation of T Cell Differentiation and Alloimmunity by the Cyclin-Dependent Kinase Inhibitor p18ink4c

PubMed Central

Rowell, Emily A.; Wang, Liqing; Chunder, Neelanjana; Hancock, Wayne W.; Wells, Andrew D.

2014-01-01

Cellular proliferation in response to mitogenic stimuli is negatively regulated by the Cip/Kip and the Ink4 families of cyclin-dependent kinase (CDK) inhibitors. Several of these proteins are elevated in anergic T cells, suggesting a potential role in the induction or maintenance of tolerance. Our previous studies showed that p27kip1 is required for the induction of T cell anergy and transplantation tolerance by costimulatory blockade, but a role for Ink4 proteins in these processes has not been established. Here we show that CD4+ T cells from mice genetically deficient for p18ink4c divide more rapidly than wild-type cells in response to antigenic, costimulatory and growth factor signals. However, this gain of proliferative function was accompanied by a moderate increase in the rate of cell death, and was accompanied by an overall defect in the generation of alloreactive IFNγ-producing effector cells. Consistent with this, p18ink4c-deficient T cells were unable to induce graft-vs-host disease in vivo, and p18ink4c deficiency cooperated with costimulatory blockade to significantly increase the survival of fully mismatched allografts in a cardiac transplantation model. While both p18ink4c and p27kip1 act to restrict T cell proliferation, p18ink4c exerts an opposite effect from p27kip1 on alloimmunity and organ transplant rejection, most likely by sustaining T cell survival and the development of effector function. Our studies point to additional important links between the cell cycle machinery and the processes of T cell differentiation, survival and tolerance. PMID:24614758

Physical Interaction of Human T-Cell Leukemia Virus Type 1 Tax with Cyclin-Dependent Kinase 4 Stimulates the Phosphorylation of Retinoblastoma Protein

PubMed Central

Haller, Kerstin; Wu, Yalin; Derow, Elisabeth; Schmitt, Iris; Jeang, Kuan-Teh; Grassmann, Ralph

2002-01-01

The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G1 phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21CIP. Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein. PMID:11971966

TOPK (T-LAK cell-originated protein kinase) inhibitor exhibits growth suppressive effect on small cell lung cancer.

PubMed

Park, Jae-Hyun; Inoue, Hiroyuki; Kato, Taigo; Zewde, Makda; Miyamoto, Takashi; Matsuo, Yo; Salgia, Ravi; Nakamura, Yusuke

2017-03-01

T-lymphokine-activated killer cell-originated protein kinase (TOPK) plays critical roles in cancer cell proliferation as well as maintenance of cancer stem cells (CSC). Small cell lung cancer (SCLC) has highly aggressive phenotype, reveals early spread to distant sites, and results in dismal prognosis with little effective treatment. In this study, we demonstrate that TOPK expression was highly upregulated in both SCLC cell lines and primary tumors. Similar to siRNA-mediated TOPK knockdown effects, treatment with a potent TOPK inhibitor, OTS514, effectively suppressed growth of SCLC cell lines (IC 50 ; 0.4-42.6 nM) and led to their apoptotic cell death. TOPK inhibition caused cell morphologic changes in SCLC cells, elongation of intercellular bridges caused by cytokinesis defects or neuronal protrusions induced by neuronal differentiation in a subset of CSC-like SCLC cells. Treatment with OTS514 suppressed forkhead box protein M1 (FOXM1) activity, which was involved in stemness of CSC. Furthermore, OTS514 treatment reduced CD90-positive SCLC cells and showed higher cytotoxic effect against lung sphere-derived CSC-like SCLC cells. Collectively, our results suggest that targeting TOPK is a promising approach for SCLC therapy. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Glycine transporters GlyT1 and GlyT2 are differentially modulated by glycogen synthase kinase 3β.

PubMed

Jiménez, Esperanza; Núñez, Enrique; Ibáñez, Ignacio; Zafra, Francisco; Aragón, Carmen; Giménez, Cecilio

2015-02-01

Inhibitory glycinergic neurotransmission is terminated by the specific glycine transporters GlyT1 and GlyT2 which actively reuptake glycine from the synaptic cleft. GlyT1 is associated with both glycinergic and glutamatergic pathways, and is the main regulator of the glycine levels in the synapses. GlyT2 is the main supplier of glycine for vesicle refilling, a process that is vital to preserve the quantal glycine content in synaptic vesicles. Therefore, to control glycinergic neurotransmission efficiently, GlyT1 and GlyT2 activity must be regulated by diverse neuronal and glial signaling pathways. In this work, we have investigated the possible functional modulation of GlyT1 and GlyT2 by glycogen synthase kinase 3 (GSK3β). This kinase is involved in mood stabilization, neurodegeneration and plasticity at excitatory and inhibitory synapses. The co-expression of GSK3β with GlyT1 or GlyT2 in COS-7 cells and Xenopus laevis oocytes, leads to inhibition and stimulation of GlyT1 and GlyT2 activities, respectively, with a decrease of GlyT1, and an increase in GlyT2 levels at the plasma membrane. The specificity of these changes is supported by the antagonism exerted by a catalytically inactive form of the kinase and through inhibitors of GSK3β such as lithium chloride and TDZD-8. GSK3β also increases the incorporation of 32Pi into GlyT1 and decreases that of GlyT2. The pharmacological inhibition of the endogenous GSK3β in neuron cultures of brainstem and spinal cord leads to an opposite modulation of GlyT1 and GlyT2.Our results suggest that GSK3β is important for stabilizing and/or controlling the expression of functional GlyTs on the neural cell surface. Copyright © 2014 Elsevier Ltd. All rights reserved.

Casein kinase 2 (CK2) increases survivin expression via enhanced β-catenin–T cell factor/lymphoid enhancer binding factor-dependent transcription

PubMed Central

Tapia, J. C.; Torres, V. A.; Rodriguez, D. A.; Leyton, L.; Quest, A. F. G.

2006-01-01

Increased expression of casein kinase 2 (CK2) is associated with hyperproliferation and suppression of apoptosis in cancer. Mutations in the tumor suppressor APC (adenomatous polyposis coli) are frequent in colon cancer and often augment β-catenin–T cell factor (Tcf)/lymphoid enhancer binding factor (Lef)-dependent transcription of genes such as c-myc and cyclin-D1. CK2 has also been implicated recently in the regulation of β-catenin stability. To identify mechanisms by which CK2 promotes survival, effects of the specific CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole were assessed. TBB and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole significantly decreased proliferation and increased apoptosis of HT29(US) colon cancer cells. RT-PCR and immunoblot analysis revealed that both inhibitors decreased survivin mRNA and protein levels in HT29(US) cells. Similar effects were observed with TBB in human DLD-1 and SW-480 colorectal cells as well as ZR-75 breast cancer cells and HEK-293T embryonic kidney cells. Expression of GFP–CK2α in HEK-293T cells resulted in β-catenin–Tcf/Lef-dependent up-regulation of survivin and increased resistance to anticancer drugs. Augmented β-catenin–Tcf/Lef-dependent transcription and resistance to apoptosis observed upon GFP–CK2α expression were abolished by TBB. Alternatively, HEK-293T cells expressing GFP–survivin were resistant to TBB-induced apoptosis. Finally, siRNA-mediated down-regulation of CK2α in HEK-293T cells coincided with reduced β-catenin and survivin levels. Taken together, these results suggest that CK2 kinase activity promotes survival by increasing survivin expression via β-catenin–Tcf/Lef-mediated transcription. Hence, selective CK2 inhibition or down-regulation in tumors may provide an attractive opportunity for the development of novel cancer therapies. PMID:17005722

Low-concentration vemurafenib induces the proliferation and invasion of human HaCaT keratinocytes through mitogen-activated protein kinase pathway activation.

PubMed

Roh, Mi Ryung; Kim, Jung Min; Lee, Sang Hee; Jang, Hong Sun; Park, Kyu Hyun; Chung, Kee Yang; Rha, Sun Young

2015-09-01

Cutaneous squamous cell carcinomas and keratoacanthomas commonly occur in patients treated with BRAF inhibitors. We investigated the effect of the BRAF inhibitor vemurafenib on normal immortalized human HaCaT keratinocytes to explore the mechanism of hyperproliferative cutaneous neoplasia associated with the use of BRAF inhibitors. Vemurafenib induced an increase in viable cell number in BRAF wild-type cell lines (SK-MEL-2 and HaCaT) but not in BRAF mutant cell lines (SK-MEL-24 and G361). In HaCaT keratinocytes, a low concentration (2 μmol/L) of vemurafenib increased cell proliferation and activated mitogen-activated protein kinase kinase/extracellular signal-regulated kinase in a CRAF-dependent manner. Invasiveness of HaCaT cells in a Matrigel assay significantly increased upon cultivation of cells with 2 μmol/L vemurafenib for 24 h. Gelatin zymography, reverse transcription polymerase chain reaction and western blot results revealed that 2 μmol/L vemurafenib treatment increased matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities in HaCaT cells. These results offer additional insight into the complex mechanism of paradoxical mitogen-activated protein kinase signaling involved in hyperproliferative cutaneous neoplasias that arise after BRAF inhibition and suggest a possible role for MMP in tumor progression and invasion. © 2015 Japanese Dermatological Association.

Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, Claire; Universite de Toulouse, UPS, IPBS, Toulouse F-31000; Lafosse, Jean-Michel

Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

Atrioventricular junction (AVJ) motion tracking: a software tool with ITK/VTK/Qt.

PubMed

Pengdong Xiao; Shuang Leng; Xiaodan Zhao; Hua Zou; Ru San Tan; Wong, Philip; Liang Zhong

2016-08-01

The quantitative measurement of the Atrioventricular Junction (AVJ) motion is an important index for ventricular functions of one cardiac cycle including systole and diastole. In this paper, a software tool that can conduct AVJ motion tracking from cardiovascular magnetic resonance (CMR) images is presented by using Insight Segmentation and Registration Toolkit (ITK), The Visualization Toolkit (VTK) and Qt. The software tool is written in C++ by using Visual Studio Community 2013 integrated development environment (IDE) containing both an editor and a Microsoft complier. The software package has been successfully implemented. From the software engineering practice, it is concluded that ITK, VTK, and Qt are very handy software systems to implement automatic image analysis functions for CMR images such as quantitative measure of motion by visual tracking.

Ibrutinib-A double-edge sword in cancer and autoimmune disorders.

PubMed

Kokhaei, Parviz; Jadidi-Niaragh, Farhad; Sotoodeh Jahromi, Abdolreza; Osterborg, Anders; Mellstedt, Håkan; Hojjat-Farsangi, Mohammad

2016-01-01

Targeted therapies have appeared as new treatment options for several disease types, including cancer and autoimmune disorders. Of several targets, tyrosine kinases (TKs) are among the most promising. Overexpression of TKs provides a target for novel therapeutic agents, including small molecule inhibitors of tyrosine kinases (TKI). Ibrutinib (PCI-32765) is a TKI of Bruton's tyrosine kinase (Btk), a key kinase of the B-cell receptor signaling pathway that plays a significant role in the proliferation, differentiation and survival of B cells. In addition to inhibitory effects, recent studies have shown that ibrutinib has multiple immunomodulatory effects. It binds covalently to IL-2 inducible tyrosine kinase (Itk) in T lymphocytes and suppresses the survival of T-helper (Th) 2 cells. This changes the balance of Th1/Th2 cells toward Th1 subset, which are the main immune cells targeting tumor cells. The dual activity of ibrutinib has paid a great attention and several studies are evaluating the anti-tumor and immunomodulatory effects in cancer, autoimmune disorders and infectious diseases. In this article we review the inhibitory and immunomodulatory effects of ibrutinib in B-cell malignancies, autoimmune diseases and infections, as well as the communication between the Ror1 receptor tyrosine kinase and BCR and effects of ibrutinib on this crosstalk.

Rapid Contour-based Segmentation for 18F-FDG PET Imaging of Lung Tumors by Using ITK-SNAP: Comparison to Expert-based Segmentation.

PubMed

Besson, Florent L; Henry, Théophraste; Meyer, Céline; Chevance, Virgile; Roblot, Victoire; Blanchet, Elise; Arnould, Victor; Grimon, Gilles; Chekroun, Malika; Mabille, Laurence; Parent, Florence; Seferian, Andrei; Bulifon, Sophie; Montani, David; Humbert, Marc; Chaumet-Riffaud, Philippe; Lebon, Vincent; Durand, Emmanuel

2018-04-03

Purpose To assess the performance of the ITK-SNAP software for fluorodeoxyglucose (FDG) positron emission tomography (PET) segmentation of complex-shaped lung tumors compared with an optimized, expert-based manual reference standard. Materials and Methods Seventy-six FDG PET images of thoracic lesions were retrospectively segmented by using ITK-SNAP software. Each tumor was manually segmented by six raters to generate an optimized reference standard by using the simultaneous truth and performance level estimate algorithm. Four raters segmented 76 FDG PET images of lung tumors twice by using ITK-SNAP active contour algorithm. Accuracy of ITK-SNAP procedure was assessed by using Dice coefficient and Hausdorff metric. Interrater and intrarater reliability were estimated by using intraclass correlation coefficients of output volumes. Finally, the ITK-SNAP procedure was compared with currently recommended PET tumor delineation methods on the basis of thresholding at 41% volume of interest (VOI; VOI 41 ) and 50% VOI (VOI 50 ) of the tumor's maximal metabolism intensity. Results Accuracy estimates for the ITK-SNAP procedure indicated a Dice coefficient of 0.83 (95% confidence interval: 0.77, 0.89) and a Hausdorff distance of 12.6 mm (95% confidence interval: 9.82, 15.32). Interrater reliability was an intraclass correlation coefficient of 0.94 (95% confidence interval: 0.91, 0.96). The intrarater reliabilities were intraclass correlation coefficients above 0.97. Finally, VOI 41 and VOI 50 accuracy metrics were as follows: Dice coefficient, 0.48 (95% confidence interval: 0.44, 0.51) and 0.34 (95% confidence interval: 0.30, 0.38), respectively, and Hausdorff distance, 25.6 mm (95% confidence interval: 21.7, 31.4) and 31.3 mm (95% confidence interval: 26.8, 38.4), respectively. Conclusion ITK-SNAP is accurate and reliable for active-contour-based segmentation of heterogeneous thoracic PET tumors. ITK-SNAP surpassed the recommended PET methods compared with ground truth

Leucine-Rich Repeat Kinase 2 Controls the Ca2+/Nuclear Factor of Activated T Cells/IL-2 Pathway during Aspergillus Non-Canonical Autophagy in Dendritic Cells.

PubMed

Wong, Alicia Yoke Wei; Oikonomou, Vasilis; Paolicelli, Giuseppe; De Luca, Antonella; Pariano, Marilena; Fric, Jan; Tay, Hock Soon; Ricciardi-Castagnoli, Paola; Zelante, Teresa

2018-01-01

The Parkinson's disease-associated protein, Leucine-rich repeat kinase 2 (LRRK2), a known negative regulator of nuclear factor of activated T cells (NFAT), is expressed in myeloid cells such as macrophages and dendritic cells (DCs) and is involved in the host immune response against pathogens. Since, the Ca 2+ /NFAT/IL-2 axis has been previously found to regulate DC response to the fungus Aspergillus , we have investigated the role played by the kinase LRRK2 during fungal infection. Mechanistically, we found that in the early stages of the non-canonical autophagic response of DCs to the germinated spores of Aspergillus , LRRK2 undergoes progressive degradation and regulates NFAT translocation from the cytoplasm to the nucleus. Our results shed new light on the complexity of the Ca 2+ /NFAT/IL-2 pathway, where LRRK2 plays a role in controlling the immune response of DCs to Aspergillus .

RAFTK, a novel member of the focal adhesion kinase family, is phosphorylated and associates with signaling molecules upon activation of mature T lymphocytes.

PubMed

Ganju, R K; Hatch, W C; Avraham, H; Ona, M A; Druker, B; Avraham, S; Groopman, J E

1997-03-17

The related adhesion focal tyrosine kinase (RAFTK), a recently discovered member of the focal adhesion kinase family, has previously been reported to participate in signal transduction in neuronal cells, megakaryocytes, and B lymphocytes. We have found that RAFTK is constitutively expressed in human T cells and is rapidly phosphorylated upon the activation of the T cell receptor (TCR). This activation also results in an increase in the autophosphorylation and kinase activity of RAFTK. After its stimulation, there was an increase in the association of the src cytoplasmic tyrosine kinase Fyn and the adapter protein Grb2. This association was mediated through the SH2 domains of Fyn and Grb2. RAFTK also co-immunoprecipitates with the SH2 domain of Lck and with the cytoskeletal protein paxillin through its COOH-terminal proline-rich domain. The tyrosine phosphorylation of RAFTK after T cell receptor-mediated stimulation was reduced by the pretreatment of cells with cytochalasin D, suggesting the role of the cytoskeleton in this process. These observations indicate that RAFTK participates in T cell receptor signaling and may act to link signals from the cell surface to the cytoskeleton and thereby affect the host immune response.

Osimertinib, a third-generation tyrosine kinase inhibitor targeting non-small cell lung cancer with EGFR T790M mutations.

PubMed

McCoach, C E; Jimeno, A

2016-10-01

Oncogenic driver mutations in the epidermal growth factor receptor (EGFR) gene have provided a focus for effective targeted therapy. Unfortunately, all patients eventually develop resistance to frontline therapy with EGFR tyrosine kinase inhibitors (TKIs). The majority of patients develop a large subclonal population of tumor cells with a T790M mutation that renders these cells resistant to first-generation TKIs. Osimertinib is a third-generation EGFR TKI that was designed to overcome resistance from T790M mutations. This agent has demonstrated strong preclinical activity, and in the clinic it has demonstrated a high objective response rate and progression-free survival in patients with EGFR double mutations (L858R/T790M and exon 19 deletion/T790M). It is now approved by the FDA for patients who have a documented T790M mutation and who have progressed on a prior TKI. Osimertinib is also approved in the E.U. and Japan. Copyright 2016 Prous Science, S.A.U. or its licensors. All rights reserved.

Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts.

PubMed

Franzolin, Elisa; Rampazzo, Chiara; Pérez-Pérez, María-Jesús; Hernández, Ana-Isabel; Balzarini, Jan; Bianchi, Vera

2006-05-26

Cellular models of mitochondrial thymidine kinase (TK2) deficiency require a reliable method to measure TK2 activity in whole cell extracts containing two interfering deoxyribonucleoside kinases, thymidine kinase 1 (TK1) and deoxycytidine kinase. We tested the value of the thymidine analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as a TK2-specific substrate. With extracts of OSTTK1- cells containing TK2 as the only thymidine kinase and a highly specific TK2 inhibitor we established conditions to detect the low TK2 activity commonly present in cells. With extracts of TK1-proficient osteosarcoma cells and normal human fibroblasts we showed that BVDU, but not 1-(beta-d-arabinofuranosyl)thymine (Ara-T), discriminates TK2 activity even in the presence of 100-fold excess TK1. A comparison with current procedures based on TK2 inhibition demonstrated the better performance of the new TK2 assay. When cultured human fibroblasts passed from proliferation to quiescence TK2 activity increased by 3-fold, stressing the importance of TK2 function in the absence of TK1.

Requirement of T-lymphokine-activated killer cell-originated protein kinase for TRAIL resistance of human HeLa cervical cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Hyeok-Ran; Lee, Ki Won; Dong, Zigang

2010-01-01

T-lymphokine-activated killer cell-originated protein kinase (TOPK) appears to be highly expressed in various cancer cells and to play an important role in maintaining proliferation of cancer cells. However, the underlying mechanism by which TOPK regulates growth of cancer cells remains elusive. Here we report that upregulated endogenous TOPK augments resistance of cancer cells to apoptosis induced by tumor necrosis factor-related apoptosis inducing ligand (TRAIL). Stable knocking down of TOPK markedly increased TRAIL-mediated apoptosis of human HeLa cervical cancer cells, as compared with control cells. Caspase 8 or caspase 3 activities in response to TRAIL were greatly incremented in TOPK-depleted cells.more » Ablation of TOPK negatively regulated TRAIL-mediated NF-{kappa}B activity. Furthermore, expression of NF-{kappa}B-dependent genes, FLICE-inhibitory protein (FLIP), inhibitor of apoptosis protein 1 (c-IAP1), or X-linked inhibitor of apoptosis protein (XIAP) was reduced in TOPK-depleted cells. Collectively, these findings demonstrated that TOPK contributed to TRAIL resistance of cancer cells via NF-{kappa}B activity, suggesting that TOPK might be a potential molecular target for successful cancer therapy using TRAIL.« less

Membrane-bound Dickkopf-1 in Foxp3+ regulatory T cells suppresses T-cell-mediated autoimmune colitis.

PubMed

Chae, Wook-Jin; Park, Jong-Hyun; Henegariu, Octavian; Yilmaz, Saliha; Hao, Liming; Bothwell, Alfred L M

2017-10-01

Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3 + regulatory T (Treg) cells use Dickkopf-1 (DKK-1) to regulate T-cell-mediated tolerance in the T-cell-mediated autoimmune colitis model. Treg cells from DKK-1 hypomorphic doubleridge mice failed to control CD4 + T-cell proliferation, resulting in CD4 T-cell-mediated autoimmune colitis. Thymus-derived Treg cells showed a robust expression of DKK-1 but not in naive or effector CD4 T cells. DKK-1 expression in Foxp3 + Treg cells was further increased upon T-cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3 + Treg cells expressed DKK-1 in the cell membrane and the functional inhibition of DKK-1 using DKK-1 monoclonal antibody abrogated the suppressor function of Foxp3 + Treg cells. DKK-1 expression was dependent on de novo protein synthesis and regulated by the mitogen-activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane-bound DKK-1 as a novel Treg-derived mediator to maintain immunological tolerance in T-cell-mediated autoimmune colitis. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

Trivanillic polyphenols with anticancer cytostatic effects through the targeting of multiple kinases and intracellular Ca2+ release

PubMed Central

Lamoral-Theys, Delphine; Wauthoz, Nathalie; Heffeter, Petra; Mathieu, Véronique; Jungwirth, Utte; Lefranc, Florence; Nève, Jean; Dubois, Jacques; Dufrasne, François; Amighi, Karim; Berger, Walter; Gailly, Philippe; Kiss, Robert

2012-01-01

Abstract Cancer cells exhibit de-regulation of multiple cellular signalling pathways and treatments of various types of cancers with polyphenols are promising. We recently reported the synthesis of a series of 33 novel divanillic and trivanillic polyphenols that displayed anticancer activity, at least in vitro, through inhibiting various kinases. This study revealed that minor chemical modifications of a trivanillate scaffold could convert cytotoxic compounds into cytostatic ones. Compound 13c, a tri-chloro derivative of trivanillic ester, displayed marked inhibitory activities against FGF-, VEGF-, EGF- and Src-related kinases, all of which are implicated not only in angiogenesis but also in the biological aggressiveness of various cancer types. The pan-anti-kinase activity of 13c occurs at less than one-tenth of its mean IC50in vitro growth inhibitory concentrations towards a panel of 12 cancer cell lines. Of the 26 kinases for which 13c inhibited their activity by >75%, eight (Yes, Fyn, FGF-R1, EGFR, Btk, Mink, Ret and Itk) are implicated in control of the actin cytoskeleton organization to varying degrees. Compound 13c accordingly impaired the typical organization of the actin cytoskeleton in human U373 glioblastoma cells. The pan-anti-kinase activity and actin cytoskeleton organization impairment provoked by 13c concomitantly occurs with calcium homeostasis impairment but without provoking MDR phenotype activation. All of these anticancer properties enabled 13c to confer therapeutic benefits in vivo in a mouse melanoma pseudometastatic lung model. These data argue in favour of further chemically modifying trivanillates to produce novel and potent anticancer drugs. PMID:21810170

Comparison of acalabrutinib, a selective Bruton tyrosine kinase inhibitor, with ibrutinib in chronic lymphocytic leukemia cells

PubMed Central

Patel, Viralkumar; Balakrishnan, Kumudha; Bibikova, Elena; Ayres, Mary; Keating, Michael J.; Wierda, William G.; Gandhi, Varsha

2017-01-01

Purpose Ibrutinib inhibits Bruton tyrosine kinase (BTK) by irreversibly binding to the Cys-481 residue in the enzyme. However, ibrutinib also inhibits several other enzymes that contain cysteine residues homologous to Cys-481 in BTK. Patients with relapsed/refractory or previously untreated chronic lymphocytic leukemia (CLL) demonstrate a high overall response rate to ibrutinib with prolonged survival. Acalabrutinib, a selective BTK inhibitor developed to minimize off-target activity, has shown promising overall response rates in patients with relapsed/refractory CLL. A head-to-head comparison of ibrutinib and acalabrutinib in CLL cell cultures and healthy T cells is needed to understand preclinical biologic and molecular effects. Experimental Design Using samples from patients with CLL, we compared the effects of both BTK inhibitors on biologic activity, chemokine production, cell migration, BTK phosphorylation, and downstream signaling in primary CLL lymphocytes and on normal T-cell signaling to determine effects on other kinases. Results Both BTK inhibitors induced modest cell death accompanied by cleavage of PARP and caspase 3. Production of CCL3 and CCL4 chemokines and pseudoemperipolesis were inhibited by both drugs to a similar degree. These drugs also showed similar inhibitory effects on phosphorylation of BTK and downstream S6 and ERK kinases. By contrast, off-target effects on SRC-family kinases were more pronounced with ibrutinib than acalabrutinib in healthy T lymphocytes. Conclusion Both BTK inhibitors show similar biological and molecular profile in primary CLL cells but appear different on their effect on normal T-cells. PMID:28034907

ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells

PubMed Central

Oliveira-Brito, Patrícia Kellen Martins; Gonçalves, Thiago Eleutério; Vendruscolo, Patrícia Edivânia; Roque-Barreira, Maria Cristina

2017-01-01

The recognition of cell surface glycans by lectins may be critical for the innate and adaptive immune responses. ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing TLR2 N-glycans and induces Th1 immunity. We recently demonstrated that ArtinM stimulated CD4+ T cells to produce proinflammatory cytokines. Here, we further studied the effects of ArtinM on adaptive immune cells. We showed that ArtinM activates murine CD4+ and CD8+ T cells, augmenting their positivity for CD25, CD69, and CD95 and showed higher interleukin (IL)-2 and interferon (IFN)-γ production. The CD4+ T cells exhibited increased T-bet expression in response to ArtinM, and IL-2 production by CD4+ and CD8+ T cells depended on the recognition of CD3εγ-chain glycans by ArtinM. The ArtinM effect on aberrantly-glycosylated neoplastic lymphocytes was studied in Jurkat T cells, in which ArtinM induced IL-2, IFN-γ, and IL-1β production, but decreased cell viability and growth. A higher frequency of AnnexinV- and propidium iodide-stained cells demonstrated the induction of Jurkat T cells apoptosis by ArtinM, and this apoptotic response was reduced by caspases and protein tyrosine kinase inhibitors. The ArtinM effects on murine T cells corroborated with the immunomodulatory property of lectin, whereas the promotion of Jurkat T cells apoptosis may reflect a potential applicability of ArtinM in novel strategies for treating lymphocytic leukemia. PMID:28665310

Pantoprazole, an FDA-approved proton-pump inhibitor, suppresses colorectal cancer growth by targeting T-cell-originated protein kinase

PubMed Central

Sun, Huimin; Xiao, Juanjuan; Lu, Tao; Huang, Guangqian; Chen, Pianpian; Zhang, Jianmin; Zhu, Feng; Li, Hua; Duan, Qiuhong

2016-01-01

T-cell-originated protein kinase (TOPK) is highly expressed in several cancer cells and promotes tumorigenesis and progression, and therefore, it is an important target for drug treatment of tumor. Pantoprazole (PPZ) was identified to be a TOPK inhibitor from FDA-approved drug database by structure based virtual ligand screening. Herein, the data indicated that pantoprazole inhibited TOPK activities by directly binding with TOPK in vitro and in vivo. Ex vivo studies showed that pantoprazole inhibited TOPK activities in JB6 Cl41 cells and HCT 116 colorectal cancer cells. Moreover, knockdown of TOPK in HCT 116 cells decreased their sensitivities to pantoprazole. Results of an in vivo study demonstrated that i.p. injection of pantoprazole in HCT 116 colon tumor-bearing mice effectively suppressed cancer growth. The TOPK downstream signaling molecule phospho-histone H3 in tumor tissues was also decreased after pantoprazole treatment. In short, pantoprazole can suppress growth of colorectal cancer cells as a TOPK inhibitor both in vitro and in vivo. PMID:26967058

Retinoic Acid Modulates Interferon-γ Production by Hepatic Natural Killer T Cells via Phosphatase 2A and the Extracellular Signal-Regulated Kinase Pathway

PubMed Central

Chang, Heng-Kwei

2015-01-01

Retinoic acid (RA), an active metabolite converted from vitamin A, plays an active role in immune function, such as defending against infections and immune regulation. Although RA affects various types of immune cells, including antigen-presenting cells, B lymphocytes, and T lymphocytes, whether it affects natural killer T (NKT) cells remain unknown. In this study, we found that RA decreased interferon (IFN)-γ production by activated NKT cells through T-cell receptor (TCR) and CD28. We also found that RA reduced extracellular signal-regulated kinase (ERK) phosphorylation, but increased phosphatase 2A (PP2A) activity in TCR/CD28-stimulated NKT cells. The increased PP2A activity, at least partly, contributed to the reduction of ERK phosphorylation. Since inhibition of ERK activation decreases IFN-γ production by TCR/CD28-stimulated NKT cells, RA may downregulate IFN-γ production by TCR/CD28-stimulated NKT cells through the PP2A-ERK pathway. Our results demonstrated a novel function of RA in modulating the IFN-γ expression by activated NKT cells. PMID:25343668

Targeting Janus tyrosine kinase 3 (JAK3) with an inhibitor induces secretion of TGF-β by CD4+ T cells

PubMed Central

Cetkovic-Cvrlje, Marina; Olson, Marin; Ghate, Ketaki

2012-01-01

Regulatory T cells (Tregs) are critical for the peripheral maintenance of the autoreactive T cells in autoimmune disorders such as type 1 diabetes (T1D). Pharmacological inhibition of Janus tyrosine kinase 3 (JAK3) has been proposed as a basis for new treatment modalities against autoimmunity and allogeneic responses. Targeting JAK3 with an inhibitor has previously been shown to exhibit protective action against the development of T1D in non-obese diabetic (NOD) mice. As the mechanism of such preventative action has been unknown, we hypothesized that JAK3 inhibition induces generation of Tregs. Here, we show that the JAK3 inhibitor 4-(4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131) suppresses proliferation of short-term cultured NOD CD4+ T cells through induction of apoptosis, while promoting survival of a particular population of long-term cultured cells. It was found that the surviving cells were not of the CD4+CD25+FoxP3+ phenotype. They secreted decreased amounts of IL-10, IL-4 and interferon (IFN)-γ compared to the cells not exposed to the optimal concentrations of JAK3 inhibitor. However, an elevated transforming growth factor (TGF)-β secretion was detected in their supernatants. In vivo treatment of prediabetic NOD mice with WHI-P131 did not affect the frequency and number of splenic and pancreatic lymph node CD4+FoxP3+ Tregs, while generating an elevated numbers of CD4+FoxP3− TGF-β-secreting T cells. In conclusion, our data suggest an induction of TGF-β-secreting CD4+ T cells as the underlying mechanism for antidiabetogenic effects obtained by the treatment with a JAK3 inhibitor. To our knowledge, this is the first report of the JAK3 inhibitor activity in the context of the murine Tregs. PMID:22728763

A winged helix forkhead (FOXD2) tunes sensitivity to cAMP in T lymphocytes through regulation of cAMP-dependent protein kinase RIalpha.

PubMed

Johansson, C Christian; Dahle, Maria K; Blomqvist, Sandra Rodrigo; Grønning, Line M; Aandahl, Einar M; Enerbäck, Sven; Taskén, Kjetil

2003-05-09

Forkhead/winged helix (FOX) transcription factors are essential for control of the cell cycle and metabolism. Here, we show that spleens from Mf2-/- (FOXD2-/-) mice have reduced mRNA (50%) and protein (35%) levels of the RIalpha subunit of the cAMP-dependent protein kinase. In T cells from Mf2-/- mice, reduced levels of RIalpha translates functionally into approximately 2-fold less sensitivity to cAMP-mediated inhibition of proliferation triggered through the T cell receptor-CD3 complex. In Jurkat T cells, FOXD2 overexpression increased the endogenous levels of RIalpha through induction of the RIalpha1b promoter. FOXD2 overexpression also increased the sensitivity of the promoter to cAMP. Finally, co-expression experiments demonstrated that protein kinase Balpha/Akt1 work together with FOXD2 to induce the RIalpha1b promoter (10-fold) and increase endogenous RIalpha protein levels further. Taken together, our data indicate that FOXD2 is a physiological regulator of the RIalpha1b promoter in vivo working synergistically with protein kinase B to induce cAMP-dependent protein kinase RIalpha expression, which increases cAMP sensitivity and sets the threshold for cAMP-mediated negative modulation of T cell activation.

Myosin 3A kinase activity is regulated by phosphorylation of the kinase domain activation loop.

PubMed

Quintero, Omar A; Unrath, William C; Stevens, Stanley M; Manor, Uri; Kachar, Bechara; Yengo, Christopher M

2013-12-27

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop*

PubMed Central

Quintero, Omar A.; Unrath, William C.; Stevens, Stanley M.; Manor, Uri; Kachar, Bechara; Yengo, Christopher M.

2013-01-01

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells. PMID:24214986

Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies.

PubMed

Friedman, Jay; Morisada, Megan; Sun, Lillian; Moore, Ellen C; Padget, Michelle; Hodge, James W; Schlom, Jeffrey; Gameiro, Sofia R; Allen, Clint T

2018-06-21

Natural killer (NK) cells recognize and lyse target tumor cells in an MHC-unrestricted fashion and complement antigen- and MHC-restricted killing by T-lymphocytes. NK cells and T-lymphocytes mediate early killing of targets through a common granzyme B-dependent mechanism. Tumor cell resistance to granzyme B and how this alters NK cell killing is not clearly defined. Tumor cell sensitivity to cultured murine KIL and human high affinity NK (haNK) cells in the presence or absence of AZD1775, a small molecule inhibitor of WEE1 kinase, was assessed via real time impedance analysis. Mechanisms of enhanced sensitivity to NK lysis were determined and in vivo validation via adoptive transfer of KIL cells into syngeneic mice was performed. Cultured murine KIL cells lyse murine oral cancer 2 (MOC2) cell targets more efficiently than freshly isolated peripheral murine NK cells. MOC2 sensitivity to granzyme B-dependent KIL cell lysis was enhanced by inhibition of WEE1 kinase, reversing G2/M cell cycle checkpoint activation and resulting in enhanced DNA damage and apoptosis. Treatment of MOC2 tumor-bearing wild-type C57BL/6 mice with AZD1775 and adoptively transferred KIL cells resulted in enhanced tumor growth control and survival over controls or either treatment alone. Validating these findings in human models, WEE1 kinase inhibition sensitized two human head and neck cancer cell lines to direct lysis by haNK cells. Further, WEE1 kinase inhibition sensitized these cell lines to antibody-dependent cell-mediated cytotoxicity when combined with the anti-PD-L1 IgG1 mAb Avelumab. Tumor cell resistance to granzyme B-induced cell death can be reversed through inhibition of WEE1 kinase as AZD1775 sensitized both murine and human head and neck cancer cells to NK lysis. These data provide the pre-clinical rationale for the combination of small molecules that reverse cell cycle checkpoint activation and NK cellular therapies.

CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling

PubMed Central

Lindquist, Jonathan A.; Arhel, Nathalie; Felder, Edward; Karl, Sabine; Haas, Tobias L.; Fulda, Simone; Walczak, Henning; Kirchhoff, Frank; Debatin, Klaus-Michael

2009-01-01

CD95 is a multifunctional receptor that induces cell death or proliferation depending on the signal, cell type, and cellular context. Here, we describe a thus far unknown function of CD95 as a silencer of T cell activation. Naive human T cells triggered by antigen-presenting cells expressing a membrane-bound form of CD95 ligand (CD95L) or stimulated by anti-CD3 and -CD28 antibodies in the presence of recombinant CD95L had reduced activation and proliferation, whereas preactivated, CD95-sensitive T cells underwent apoptosis. Triggering of CD95 during T cell priming interfered with proximal T cell receptor signaling by inhibiting the recruitment of ζ-chain–associated protein of 70 kD, phospholipase-γ, and protein kinase C-θ into lipid rafts, thereby preventing their mutual tyrosine protein phosphorylation. Subsequently, Ca2+ mobilization and nuclear translocation of transcription factors NFAT, AP1, and NF-κB were strongly reduced, leading to impaired cytokine secretion. CD95-mediated inhibition of proliferation in naive T cells could not be reverted by the addition of exogenous interleukin-2 and T cells primed by CD95 co-stimulation remained partially unresponsive upon secondary T cell stimulation. HIV infection induced CD95L expression in primary human antigeen-presenting cells, and thereby suppressed T cell activation, suggesting that CD95/CD95L-mediated silencing of T cell activation represents a novel mechanism of immune evasion. PMID:19487421

delta opioid receptors stimulate Akt-dependent phosphorylation of c-jun in T cells.

PubMed

Shahabi, Nahid A; McAllen, Kathy; Sharp, Burt M

2006-02-01

Activation of naive T cells markedly up-regulates the expression of delta opioid receptors (DORs). These receptors are bound by DOR peptides released by T cells, modulating T cell functions such as interleukin-2 production, cellular proliferation, and chemotaxis. Previous studies have shown that DOR agonists [e.g., [D-Ala(2)-D-Leu(5)]-enkephalin (DADLE)] modulate T cell antigen receptor signaling through mitogen-activated protein kinases (MAPKs; i.e., extracellular signal-regulated kinases 1 and 2) and that DORs directly induce phosphorylation of activating transcription factor-2 (implicated in cytokine gene transcription) and its association with the MAPK c-jun1 NH(2)-terminal kinase (JNK). Such observations suggest that DORs may induce the phosphorylation of c-jun. These experiments were performed to test this hypothesis and determine the potential roles of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B). DADLE (10(-10) to 10(-6) M) dose-dependently induced c-jun phosphorylation. This was blocked by pertussis toxin and the DOR-specific antagonist naltindole. Fluorescence flow cytometry showed that DADLE significantly stimulated c-jun phosphorylation by T cells. DADLE stimulated phosphorylation of membrane-associated Akt; wortmannin and LY294002 ([2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]), specific inhibitors of PI3K, abolished the DADLE-induced phosphorylation of c-jun. Finally, inhibitors of Akt and JNK blocked DADLE-induced phosphorylation of c-jun. Thus, activated DORs directly stimulate c-jun phosphorylation through a PI3K-dependent pathway in T cells, apparently involving Akt. This implies that DORs activate JNK through a novel pathway dependent on PI3K and Akt, thereby regulating the function of activator protein-1 transcription complexes containing c-jun and other transcription partners.

The kinase DYRK1A reciprocally regulates the differentiation of Th17 and regulatory T cells

PubMed Central

Khor, Bernard; Gagnon, John D; Goel, Gautam; Roche, Marly I; Conway, Kara L; Tran, Khoa; Aldrich, Leslie N; Sundberg, Thomas B; Paterson, Alison M; Mordecai, Scott; Dombkowski, David; Schirmer, Melanie; Tan, Pauline H; Bhan, Atul K; Roychoudhuri, Rahul; Restifo, Nicholas P; O'Shea, John J; Medoff, Benjamin D; Shamji, Alykhan F; Schreiber, Stuart L; Sharpe, Arlene H; Shaw, Stanley Y; Xavier, Ramnik J

2015-01-01

The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity. DOI: http://dx.doi.org/10.7554/eLife.05920.001 PMID:25998054

Meriolins, a new class of cell death inducing kinase inhibitors with enhanced selectivity for cyclin-dependent kinases.

PubMed

Bettayeb, Karima; Tirado, Oscar M; Marionneau-Lambot, Séverine; Ferandin, Yoan; Lozach, Olivier; Morris, Jonathan C; Mateo-Lozano, Silvia; Drueckes, Peter; Schächtele, Christoph; Kubbutat, Michael H G; Liger, François; Marquet, Bernard; Joseph, Benoît; Echalier, Aude; Endicott, Jane A; Notario, Vicente; Meijer, Laurent

2007-09-01

Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.

Akt-RSK-S6-kinase Signaling Networks Activated by Oncogenic Receptor Tyrosine Kinases

PubMed Central

Moritz, Albrecht; Li, Yu; Guo, Ailan; Villén, Judit; Wang, Yi; MacNeill, Joan; Kornhauser, Jon; Sprott, Kam; Zhou, Jing; Possemato, Anthony; Ren, Jian Min; Hornbeck, Peter; Cantley, Lewis C.; Gygi, Steven P.; Rush, John; Comb, Michael J.

2011-01-01

Receptor tyrosine kinases (RTKs) activate pathways mediated by serine/threonine (Ser/Thr) kinases such as the PI3K (phosphatidylinositol 3-kinase)-Akt pathway, the Ras-MAPK (mitogen-activated protein kinase)-RSK pathway, and the mTOR (mammalian target of rapamycin)-p70 S6 pathway that control important aspects of cell growth, proliferation, and survival. The Akt, RSK, and p70 S6 family of protein kinases transmit signals by phosphorylating substrates on a RxRxxS/T motif. Here, we developed a large-scale proteomic approach to identify over 200 substrates of this kinase family in cancer cell lines driven by the c-Met, epidermal growth factor receptor (EGFR), or platelet-derived growth factor receptor a (PDGFRα) RTKs. We identified a subset of proteins with RxRxxS/T sites for which phosphorylation was decreased by RTKIs as well as by inhibitors of the PI3K, mTOR, and MAPK pathways and determined the effects of siRNA directed against these substrates on cell viability. We found that phosphorylation of the protein chaperone SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) at Ser305 is essential for PDGFRα stabilization and cell survival in PDGFRα-dependent cancer cells. Our approach provides a new view of RTK and Akt-RSK-S6 kinase signaling, revealing many previously unidentified Akt-RSK-S6 kinase substrates that merit further consideration as targets for combination therapy with RTKIs. PMID:20736484

Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation.

PubMed

Celada, Lindsay J; Rotsinger, Joseph E; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene; Drake, Wonder P

2017-01-01

Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4 + T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4 + T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1 + CD4 + T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = -0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4 + T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression.

Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation

PubMed Central

Celada, Lindsay J.; Rotsinger, Joseph E.; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene

2017-01-01

Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4+ T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4+ T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1+ CD4+ T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = −0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4+ T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression. PMID:27564547

Bruton’s Tyrosine Kinase Promotes Persistence of Mature Anti-Insulin B Cells

PubMed Central

Bonami, Rachel H.; Sullivan, Allison M.; Case, James B.; Steinberg, Hannah E.; Hoek, Kristen L.; Khan, Wasif N.; Kendall, Peggy L.

2014-01-01

Autoreactive B lymphocytes are essential for the development of T cell–mediated type 1 diabetes (T1D). Cytoplasmic Bruton’s tyrosine kinase (BTK) is a key component of B cell signaling, and its deletion in T1D-prone NOD mice significantly reduces diabetes. However, the role of BTK in the survival and function of autoreactive B cells is not clear. To evaluate the contributions of BTK, we used mice in which B cells express an anti-insulin BCR (125Tg) and promote T1D, despite being anergic. Crossing Btk deficiency onto 125Tg mice reveals that, in contrast to immature B cells, mature anti-insulin B cells are exquisitely dependent upon BTK, because their numbers are reduced by 95%. BTK kinase domain inhibition reproduces this effect in mature anti-insulin B cells, with less impact at transitional stages. The increased dependence of anti-insulin B cells on BTK became particularly evident in an Igκ locus site–directed model, in which 50% of B cells edit their BCRs to noninsulin specificities; Btk deficiency preferentially depletes insulin binders from the follicular and marginal zone B cell subsets. The persistent few Btk-deficient anti-insulin B cells remain competent to internalize Ag and invade pancreatic islets. As such, loss of BTK does not significantly reduce diabetes incidence in 125Tg/NOD mice as it does in NOD mice with a normal B cell repertoire. Thus, BTK targeting may not impair autoreactive anti-insulin B cell function, yet it may provide protection in an endogenous repertoire by decreasing the relative availability of mature autoreactive B cells. PMID:24453243

Changes in T-cell subpopulations and cytokine network during early period of ibrutinib therapy in chronic lymphocytic leukemia patients: the significant decrease in T regulatory cells number.

PubMed

Podhorecka, Monika; Goracy, Aneta; Szymczyk, Agnieszka; Kowal, Malgorzata; Ibanez, Blanca; Jankowska-Lecka, Olga; Macheta, Arkadiusz; Nowaczynska, Aleksandra; Drab-Urbanek, Elzbieta; Chocholska, Sylwia; Jawniak, Dariusz; Hus, Marek

2017-05-23

B cell receptor (BCR) stimulation signal plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL), and kinase inhibitors directed toward the BCR pathway are now the promising anti-leukemic drugs. Ibrutinib, a Bruton tyrosine kinase inhibitor, demonstrates promising clinical activity in CLL. It is reported that ibrutinib, additionally to directly targeting leukemic cells, also inhibits the interactions of these cells with T cells, macrophages and accessory cells. Assessment of these mechanisms is important because of their non -direct anti-leukemic effects and to identify possible side effects connected with long-term drug administration.The aim of this study was to assess the in vivo effects of ibrutinib on T-cell subpopulations and cytokine network in CLL. The analysis was performed on a group of 19 patients during first month of ibrutinib therapy. The standard multicolor flow cytometry and cytometric bead array methods were used for assessment of T-cell subsets and cytokines/chemokines, respectively.The data obtained indicates that Ibrutinib treatment results in changes in T-cell subpopulations and cytokine network in CLL patients. Particularly, a significant reduction of T regulatory cells in peripheral blood was observed. By targeting these populations of T cells Ibrutinib can stimulate rejection of tumor cells by the immune system.

Allosteric Fine-Tuning of the Binding Pocket Dynamics in the ITK SH2 Domain by a Distal Molecular Switch: An Atomistic Perspective.

PubMed

Momin, Mohamed; Xin, Yao; Hamelberg, Donald

2017-06-29

Although the regulation of function of proteins by allosteric interactions has been identified in many subcellular processes, molecular switches are also known to induce long-range conformational changes in proteins. A less well understood molecular switch involving cis-trans isomerization of a peptidyl-prolyl bond could induce a conformational change directly to the backbone that is propagated to other parts of the protein. However, these switches are elusive and hard to identify because they are intrinsic to biomolecules that are inherently dynamic. Here, we explore the conformational dynamics and free energy landscape of the SH2 domain of interleukin-2-inducible T-cell or tyrosine kinase (ITK) to fully understand the conformational coupling between the distal cis-trans molecular switch and its binding pocket of the phosphotyrosine motif. We use multiple microsecond-long all-atom molecular dynamics simulations in explicit water for over a total of 60 μs. We show that cis-trans isomerization of the Asn286-Pro287 peptidyl-prolyl bond is directly coupled to the dynamics of the binding pocket of the phosphotyrosine motif, in agreement with previous NMR experiments. Unlike the cis state that is localized and less dynamic in a single free energy basin, the trans state samples two distinct conformations of the binding pocket-one that recognizes the phosphotyrosine motif and the other that is somewhat similar to that of the cis state. The results provide an atomic-level description of a less well understood allosteric regulation by a peptidyl-prolyl cis-trans molecular switch that could aid in the understanding of normal and aberrant subcellular processes and the identification of these elusive molecular switches in other proteins.

Interactive deformation registration of endorectal prostate MRI using ITK thin plate splines.

PubMed

Cheung, M Rex; Krishnan, Karthik

2009-03-01

Magnetic resonance imaging with an endorectal coil allows high-resolution imaging of prostate cancer and the surrounding normal organs. These anatomic details can be used to direct radiotherapy. However, organ deformation introduced by the endorectal coil makes it difficult to register magnetic resonance images for treatment planning. In this study, plug-ins for the volume visualization software VolView were implemented on the basis of algorithms from the National Library of Medicine's Insight Segmentation and Registration Toolkit (ITK). Magnetic resonance images of a phantom simulating human pelvic structures were obtained with and without the endorectal coil balloon inflated. The prostate not deformed by the endorectal balloon was registered to the deformed prostate using an ITK thin plate spline (TPS). This plug-in allows the use of crop planes to limit the deformable registration in the region of interest around the prostate. These crop planes restricted the support of the TPS to the area around the prostate, where most of the deformation occurred. The region outside the crop planes was anchored by grid points. The TPS was more accurate in registering the local deformation of the prostate compared with a TPS variant, the elastic body spline. The TPS was also applied to register an in vivo T(2)-weighted endorectal magnetic resonance image. The intraprostatic tumor was accurately registered. This could potentially guide the boosting of intraprostatic targets. The source and target landmarks were placed graphically. This TPS plug-in allows the registration to be undone. The landmarks could be added, removed, and adjusted in real time and in three dimensions between repeated registrations. This interactive TPS plug-in allows a user to obtain a high level of accuracy satisfactory to a specific application efficiently. Because it is open-source software, the imaging community will be able to validate and improve the algorithm.

δ-Tocopherol inhibits receptor tyrosine kinase-induced AKT activation in prostate cancer cells.

PubMed

Wang, Hong; Hong, Jungil; Yang, Chung S

2016-11-01

The cancer preventive activity of vitamin E is suggested by epidemiological studies and supported by animal studies with vitamin E forms, γ-tocopherol and δ-tocopherol (δ-T). Several recent large-scale cancer prevention trials with high dose of α-tocopherol, however, yielded disappointing results. Whether vitamin E prevents or promotes cancer is a serious concern. A better understanding of the molecular mechanisms of action of the different forms of tocopherols would enhance our understanding of this topic. In this study, we demonstrated that δ-T was the most effective tocopherol form in inhibiting prostate cancer cell growth, by inducing cell cycle arrest and apoptosis. By profiling the effects of δ-T on the cell signaling using the phospho-kinase array, we found that the most inhibited target was the phosphorylation of AKT on T308. Further study on the activation of AKT by EGFR and IGFR revealed that δ-T attenuated the EGF/IGF-induced activation of AKT (via the phosphorylation of AKT on T308 induced by the activation of PIK3). Expression of dominant active PIK3 and AKT in prostate cancer cell line DU145 in which PIK3, AKT, and PTEN are wild type caused the cells to be reflectory to the inhibition of δ-T, supporting that δ-T inhibits the PIK3-mediated activation of AKT. Our data also suggest that δ-T interferes with the EGF-induced EGFR internalization, which leads to the inhibition of the receptor tyrosine kinase-dependent activation of AKT. In summary, our results revealed a novel mechanism of δ-T in inhibiting prostate cancer cell growth, supporting the cancer preventive activity δ-T. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Cyclin-dependent kinase 9 is a novel specific molecular target in adult T-cell leukemia/lymphoma.

PubMed

Narita, Tomoko; Ishida, Takashi; Ito, Asahi; Masaki, Ayako; Kinoshita, Shiori; Suzuki, Susumu; Takino, Hisashi; Yoshida, Takashi; Ri, Masaki; Kusumoto, Shigeru; Komatsu, Hirokazu; Imada, Kazunori; Tanaka, Yuetsu; Takaori-Kondo, Akifumi; Inagaki, Hiroshi; Scholz, Arne; Lienau, Philip; Kuroda, Taruho; Ueda, Ryuzo; Iida, Shinsuke

2017-08-31

Cyclin-dependent kinase 9 (CDK9), a subunit of the positive transcription elongation factor b (P-TEFb) complex, regulates gene transcription elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). The deregulation of CDK9/P-TEFb has important implications for many cancer types. BAY 1143572 is a novel and highly selective CDK9/P-TEFb inhibitor currently being investigated in phase 1 studies. We evaluated the therapeutic potential of BAY 1143572 in adult T-cell leukemia/lymphoma (ATL). As a result of CDK9 inhibition and subsequent inhibition of phosphorylation at serine 2 of the RNAPII CTD, BAY 1143572 decreased c-Myc and Mcl-1 levels in ATL-derived or human T-cell lymphotropic virus type-1 (HTLV-1)-transformed lines and primary ATL cells tested, leading to their growth inhibition and apoptosis. Median inhibitory concentrations for BAY 1143572 in ATL-derived or HTLV-1-transformed lines (n = 8), primary ATL cells (n = 11), and CD4 + cells from healthy volunteers (n = 5) were 0.535, 0.30, and 0.36 μM, respectively. Next, NOG mice were used as recipients of tumor cells from an ATL patient. BAY 1143572-treated ATL-bearing mice (once daily 12.5 mg/kg oral application) demonstrated significantly decreased ATL cell infiltration of the liver and bone marrow, as well as decreased human soluble interleukin-2 receptor levels in serum (reflecting the ATL tumor burden), compared with untreated mice (n = 8 for both). BAY 1143572-treated ATL-bearing mice demonstrated significantly prolonged survival compared with untreated ATL-bearing mice (n = 7 for both). Collectively, this study indicates that BAY 1143572 showed strong potential as a novel treatment of ATL. © 2017 by The American Society of Hematology.

Aurora kinase A revives dormant laryngeal squamous cell carcinoma cells via FAK/PI3K/Akt pathway activation

PubMed Central

Yang, Li-yun; He, Chang-yu; Chen, Xue-hua; Su, Li-ping; Liu, Bing-ya; Zhang, Hao

2016-01-01

Revival of dormant tumor cells may be an important tumor metastasis mechanism. We hypothesized that aurora kinase A (AURKA), a cell cycle control kinase, promotes the transition of laryngeal squamous cell carcinoma (LSCC) cells from G0 phase to active division. We therefore investigated whether AURKA could revive dormant tumor cells to promote metastasis. Western blotting revealed that AURKA expression was persistently low in dormant laryngeal cancer Hep2 (D-Hep2) cells and high in non-dormant (T-Hep2) cells. Decreasing AURKA expression in T-Hep2 cells induced dormancy and reduced FAK/PI3K/Akt pathway activity. Increasing AURKA expression in D-Hep2 cells increased FAK/PI3K/Akt pathway activity and enhanced cellular proliferation, migration, invasion and metastasis. In addition, FAK/PI3K/Akt pathway inhibition caused dormancy-like behavior and reduced cellular mobility, migration and invasion. We conclude that AURKA may revive dormant tumor cells via FAK/PI3K/Akt pathway activation, thereby promoting migration and invasion in laryngeal cancer. AURKA/FAK/PI3K/Akt inhibitors may thus represent potential targets for clinical LSCC treatment. PMID:27356739

T-Cell-Specific Loss of the PI-3-Kinase p110α Catalytic Subunit Results in Enhanced Cytokine Production and Antitumor Response

PubMed Central

Aragoneses-Fenoll, Laura; Ojeda, Gloria; Montes-Casado, María; Acosta-Ampudia, Yeny; Dianzani, Umberto; Portolés, Pilar; Rojo, José M.

2018-01-01

Class IA phosphatidylinositol 3-kinase (PI3K) catalytic subunits p110α and p110δ are targets in cancer therapy expressed at high levels in T lymphocytes. The role of p110δ PI3K in normal or pathological immune responses is well established, yet the importance of p110α subunits in T cell-dependent immune responses is not clear. To address this problem, mice with p110α conditionally deleted in CD4+ and CD8+ T lymphocytes (p110α−/−ΔT) were used. p110α−/−ΔT mice show normal development of T cell subsets, but slightly reduced numbers of CD4+ T cells in the spleen. “In vitro,” TCR/CD3 plus CD28 activation of naive CD4+ and CD8+ p110α−/−ΔT T cells showed enhanced effector function, particularly IFN-γ secretion, T-bet induction, and Akt, Erk, or P38 activation. Tfh derived from p110α−/−ΔT cells also have enhanced responses when compared to normal mice, and IL-2 expanded p110α−/−ΔT CD8+ T cells had enhanced levels of LAMP-1 and Granzyme B. By contrast, the expansion of p110α−/−ΔT iTreg cells was diminished. Also, p110α−/−ΔT mice had enhanced anti-keyhole limpet hemocyanin (KLH) IFN-γ, or IL-4 responses and IgG1 and IgG2b anti-KLH antibodies, using CFA or Alum as adjuvant, respectively. When compared to WT mice, p110α−/−ΔT mice inoculated with B16.F10 melanoma showed delayed tumor progression. The percentage of CD8+ T lymphocytes was higher and the percentage of Treg cells lower in the spleen of tumor-bearing p110α−/−ΔT mice. Also, IFN-γ production in tumor antigen-activated spleen cells was enhanced. Thus, PI3K p110α plays a significant role in antigen activation and differentiation of CD4+ and CD8+ T lymphocytes modulating antitumor immunity. PMID:29535720

Overactivation of Mitogen-Activated Protein Kinase and Suppression of Mitofusin-2 Expression Are Two Independent Events in High Mobility Group Box 1 Protein–Mediated T Cell Immune Dysfunction

PubMed Central

Tang, Lu-ming; Zhao, Guang-ju; Zhu, Xiao-mei; Dong, Ning; Yu, Yan

2013-01-01

High mobility group box 1 protein (HMGB1), a critical proinflammatory cytokine, has recently been identified to be an immunostimulatory signal involved in sepsis-related immune dysfunction when released extracellularly, but the potential mechanism involved remains elusive. Here, we showed that the treatment with HMGB1 in vitro inhibited T lymphocyte immune response and expression of mitofusin-2 (Mfn-2; a member of the mitofusin family) in a dose- and time-dependent manner. Upregulation of Mfn-2 expression attenuated the suppressive effect of HMGB1 on T cell immune function. The phosphorylation of both extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) was markedly upregulated by treating with high amount of HMGB1, while pretreatment with ERK1/2 and p38 MAPK-specific inhibitors (U0126 and SB203580) could attenuate suppression of T cell immune function and nuclear factor of activated T cell (NFAT) activation induced by HMGB1, respectively. HMGB1-induced activity of ERK1/2 and p38 was not fully inhibited in the presence of U0126 or SB203580. Interestingly, overexpression of Mfn-2 had no marked effect on HMGB1-mediated activation of MAPK, but could attenuate the suppressive effect of HMGB1 on the activity of NFAT. Thus, the mechanisms involved in HMGB1-induced T cell immune dysfunction in vitro at least partly include suppression of Mfn-2 expression, overactivation of ERK1/2, p38 MAPK, and intervention of NFAT activation, while the protective effect of Mfn-2 on T cell immune dysfunction induced by HMGB1 is dependent on other signaling pathway associated with NFAT, but not MAPK. Taken together, we conclude that overactivation of MAPK and suppression of Mfn-2 expression are two independent events in HMGB1-mediated T cell immune dysfunction. PMID:23697559

Truncated tyrosine kinase B brain-derived neurotrophic factor receptor directs cortical neural stem cells to a glial cell fate by a novel signaling mechanism.

PubMed

Cheng, Aiwu; Coksaygan, Turhan; Tang, Hongyan; Khatri, Rina; Balice-Gordon, Rita J; Rao, Mahendra S; Mattson, Mark P

2007-03-01

During development of the mammalian cerebral cortex neural stem cells (NSC) first generate neurons and subsequently produce glial cells. The mechanism(s) responsible for this developmental shift from neurogenesis to gliogenesis is unknown. Brain-derived neurotrophic factor (BDNF) is believed to play important roles in the development of the mammalian cerebral cortex; it enhances neurogenesis and promotes the differentiation and survival of newly generated neurons. Here, we provide evidence that a truncated form of the BDNF receptor tyrosine kinase B (trkB-t) plays a pivotal role in directing embryonic mouse cortical NSC to a glial cell fate. Expression of trkB-t promotes differentiation of NSC toward astrocytes while inhibiting neurogenesis both in cell culture and in vivo. The mechanism by which trkB-t induces astrocyte genesis is not simply the result of inhibition of full-length receptor with intrinsic tyrosine kinase activity signaling. Instead, binding of BDNF to trkB-t activates a signaling pathway (involving a G-protein and protein kinase C) that induced NSC to become glial progenitors and astrocytes. Thus, the increased expression of trkB-t in the embryonic cerebral cortex that occurs coincident with astrocyte production plays a pivotal role in the developmental transition from neurogenesis to gliogenesis. Our findings suggest a mechanism by which a single factor (BDNF) regulates the production of the two major cell types in the mammalian cerebral cortex.

BCL11B enhances TCR/CD28-triggered NF-kappaB activation through up-regulation of Cot kinase gene expression in T-lymphocytes.

PubMed

Cismasiu, Valeriu B; Duque, Javier; Paskaleva, Elena; Califano, Danielle; Ghanta, Sailaja; Young, Howard A; Avram, Dorina

2009-01-15

BCL11B is a transcriptional regulator with an important role in T-cell development and leukaemogenesis. We demonstrated recently that BCL11B controls expression from the IL (interleukin)-2 promoter through direct binding to the US1 (upstream site 1). In the present study, we provide evidence that BCL11B also participates in the activation of IL-2 gene expression by enhancing NF-kappaB (nuclear factor kappaB) activity in the context of TCR (T-cell receptor)/CD28-triggered T-cell activation. Enhanced NF-kappaB activation is not a consequence of BCL11B binding to the NF-kappaB response elements or association with the NF-kappaB-DNA complexes, but rather the result of higher translocation of NF-kappaB to the nucleus caused by enhanced degradation of IkappaB (inhibitor of NF-kappaB). The enhanced IkappaB degradation in cells with increased levels of BCL11B was specific for T-cells activated through the TCR, but not for cells activated through TNFalpha (tumour necrosis factor alpha) or UV light, and was caused by increased activity of IkappaB kinase, as indicated by its increase in phosphorylation. As BCL11B is a transcription factor, we investigated whether the expression of genes upstream of IkappaB kinase in the TCR/CD28 signalling pathway was affected by increased BCL11B expression, and found that Cot (cancer Osaka thyroid oncogene) kinase mRNA levels were elevated. Cot kinase is known to promote enhanced IkappaB kinase activity, which results in the phosphorylation and degradation of IkappaB and activation of NF-kappaB. The implied involvement of Cot kinase in BCL11B-mediated NF-kappaB activation in response to TCR activation is supported by the fact that a Cot kinase dominant-negative mutant or Cot kinase siRNA (small interfering RNA) knockdown blocked BCL11B-mediated NF-kappaB activation. In support of our observations, in the present study we report that BCL11B enhances the expression of several other NF-kappaB target genes, in addition to IL-2. In addition, we

Selective Effects of PD-1 on Akt and Ras Pathways Regulate Molecular Components of the Cell Cycle and Inhibit T Cell Proliferation

PubMed Central

Patsoukis, Nikolaos; Brown, Julia; Petkova, Victoria; Liu, Fang; Li, Lequn; Boussiotis, Vassiliki A.

2017-01-01

The receptor programmed death 1 (PD-1) inhibits T cell proliferation and plays a critical role in suppressing self-reactive T cells, and it also compromises antiviral and antitumor responses. To determine how PD-1 signaling inhibits T cell proliferation, we used human CD4+ T cells to examine the effects of PD-1 signaling on the molecular control of the cell cycle. The ubiquitin ligase SCFSkp2 degrades p27kip1, an inhibitor of cyclin-dependent kinases (Cdks), and PD-1 blocked cell cycle progression through the G1 phase by suppressing transcription of SKP2, which encodes a component of this ubiquitin ligase. Thus, in T cells stimulated through PD-1, Cdks were not activated, and two critical Cdk substrates were not phosphorylated. Activation of PD-1 inhibited phosphorylation of the retinoblastoma gene product, which suppressed expression of E2F target genes. PD-1 also inhibited phosphorylation of the transcription factor Smad3, which increased its activity. These events induced additional inhibitory checkpoints in the cell cycle by increasing the abundance of the G1 phase inhibitor p15INK4 and repressing the Cdk-activating phosphatase Cdc25A. PD-1 suppressed SKP2 transcription by inhibiting phosphoinositide 3-kinase–Akt and Ras–mitogen-activated and extracellular signal–regulated kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) signaling. Exposure of cells to the proliferation-promoting cytokine interleukin-2 restored activation of MEK-ERK signaling, but not Akt signaling, and only partially restored SKP2 expression. Thus, PD-1 blocks cell cycle progression and proliferation of T lymphocytes by affecting multiple regulators of the cell cycle. PMID:22740686

Protein kinase D1 phosphorylates HDAC7 and induces its nuclear export after T-cell receptor activation.

PubMed

Parra, Maribel; Kasler, Herbert; McKinsey, Timothy A; Olson, Eric N; Verdin, Eric

2005-04-08

HDAC7, a class II histone deacetylase that is highly expressed in thymocytes, inhibits both transcription of the orphan steroid nuclear receptor Nur77 and induction of apoptosis in response to activation of the T-cell receptor (TCR). Here, we report that HDAC7 is exported to the cytoplasm by a calcium-independent signaling pathway after TCR activation. Protein kinase D1 (PKD1) was activated after TCR engagement, interacted with HDAC7, and phosphorylated three serines (Ser155, Ser318, and Ser448) at its N terminus, leading to its export from the nucleus. Mutation of Ser155, Ser318, and Ser448 blocked the nucleocytoplasmic shuttling of HDAC7 in response to TCR activation, as did overexpression of a kinase-inactive form of PKD1. Consistent with the regulatory role of HDAC7 in Nur77 expression, PKD1 activation led to the transcriptional activation of Nur77 via myocyte enhancer factor 2-binding sites in its promoter. In a mouse model of negative selection, PKD1 was activated during thymocyte activation. These observations indicate that PKD1 regulates the expression of Nur77 during thymocyte activation at least in part by phosphorylating HDAC7.

Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling

PubMed Central

Linnemann, Carsten; Schildberg, Frank A; Schurich, Anna; Diehl, Linda; Hegenbarth, Silke I; Endl, Elmar; Lacher, Svenja; Müller, Christa E; Frey, Jürgen; Simeoni, Luca; Schraven, Burkhart; Stabenow, Dirk; Knolle, Percy A

2009-01-01

Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after αCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve αCD3/CD28-stimulated CD8 cells. Consequently, αCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs. PMID:19740334

Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling.

PubMed

Linnemann, Carsten; Schildberg, Frank A; Schurich, Anna; Diehl, Linda; Hegenbarth, Silke I; Endl, Elmar; Lacher, Svenja; Müller, Christa E; Frey, Jürgen; Simeoni, Luca; Schraven, Burkhart; Stabenow, Dirk; Knolle, Percy A

2009-09-01

Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after alphaCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve alphaCD3/CD28-stimulated CD8 cells. Consequently, alphaCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs.

Identification and characterization of ALK kinase splicing isoforms in non-small-cell lung cancer

PubMed Central

de Figueiredo-Pontes, Lorena Lobo; Wong, Daisy Wing-Sze; Tin, Vick Pui-Chi; Chung, Lap-Ping; Yasuda, Hiroyuki; Yamaguchi, Norihiro; Nakayama, Sohei; Jänne, Pasi Antero; Wong, Maria Pik; Kobayashi, Susumu Soeda; Costa, Daniel Botelho

2014-01-01

Purpose: Anaplastic lymphoma kinase (ALK) rearrangements are present in an important subset of non-small-cell lung cancer (NSCLC) and predict for response to the tyrosine kinase inhibitor crizotinib. In this study, we evaluated the yet unknown frequency and functional role of ALK splicing isoforms in NSCLC. Experimental Design: We analyzed 270 cases of NSCLC for ALK kinase domain splicing aberrations, and in addition generated constructs with full length EML4-ALK (E13;A20) and a splicing isoform. Results: Splicing isoforms of the kinase domain of ALK - including complete skipping of exon 23 (ALKdel23, ALK p.I1171fs*42) and exon 27 (ALKdel27, ALK p.T1312fs*0) - were identified in 11.1% (30/270 cases) of NSCLC, and these changes co-existed with ALK rearrangements, KRAS mutations and EGFR mutations. ALK splicing isoforms were observed with full length EML4-ALK in crizotinib-naïve and treated NSCLCs. ALK T1312fs*0 was unable to render cells solely dependent on ALK signaling. Unlike EML4-ALK and EML4-ALK p.L1196M, EML4-ALK T1312fs*0 did not autophosphorylate ALK or other phospho-tyrosine sites. Co-expression of equal amounts of EML4-ALK T1312fs*0 and EML4-ALK did not result in resistance to crizotinib, while co-expression of EML4-ALK L1196M with EML4-ALK resulted in resistance to inhibition of ALK by crizotinib. Conclusions: ALK kinase splicing isoforms were present in NSCLC and even if translated seemed to be non-functional variants of ALK. PMID:24419423

Dynamics of the Tec‐family tyrosine kinase SH3 domains

PubMed Central

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

2016-01-01

Abstract 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. PMID:26808198

Membrane Transfer from Mononuclear Cells to Polymorphonuclear Neutrophils Transduces Cell Survival and Activation Signals in the Recipient Cells via Anti-Extrinsic Apoptotic and MAP Kinase Signaling Pathways.

PubMed

Li, Ko-Jen; Wu, Cheng-Han; Shen, Chieh-Yu; Kuo, Yu-Min; Yu, Chia-Li; Hsieh, Song-Chou

2016-01-01

The biological significance of membrane transfer (trogocytosis) between polymorphonuclear neutrophils (PMNs) and mononuclear cells (MNCs) remains unclear. We investigated the biological/immunological effects and molecular basis of trogocytosis among various immune cells in healthy individuals and patients with active systemic lupus erythematosus (SLE). By flow cytometry, we determined that molecules in the immunological synapse, including HLA class-I and-II, CD11b and LFA-1, along with CXCR1, are exchanged among autologous PMNs, CD4+ T cells, and U937 cells (monocytes) after cell-cell contact. Small interfering RNA knockdown of the integrin adhesion molecule CD11a in U937 unexpectedly enhanced the level of total membrane transfer from U937 to PMN cells. Functionally, phagocytosis and IL-8 production by PMNs were enhanced after co-culture with T cells. Total membrane transfer from CD4+ T to PMNs delayed PMN apoptosis by suppressing the extrinsic apoptotic molecules, BAX, MYC and caspase 8. This enhancement of activities of PMNs by T cells was found to be mediated via p38- and P44/42-Akt-MAP kinase pathways and inhibited by the actin-polymerization inhibitor, latrunculin B, the clathrin inhibitor, Pitstop-2, and human immunoglobulin G, but not by the caveolin inhibitor, methyl-β-cyclodextrin. In addition, membrane transfer from PMNs enhanced IL-2 production by recipient anti-CD3/anti-CD28 activated MNCs, and this was suppressed by inhibitors of mitogen-activated protein kinase (PD98059) and protein kinase C (Rottlerin). Of clinical significance, decreased total membrane transfer from PMNs to MNCs in patients with active SLE suppressed mononuclear IL-2 production. In conclusion, membrane transfer from MNCs to PMNs, mainly at the immunological synapse, transduces survival and activation signals to enhance PMN functions and is dependent on actin polymerization, clathrin activation, and Fcγ receptors, while membrane transfer from PMNs to MNCs depends on MAP kinase and

Ibrutinib efficacy and tolerability in patients with relapsed chronic lymphocytic leukemia following allogeneic HCT

PubMed Central

Ryan, Christine E.; Sahaf, Bita; Logan, Aaron C.; O’Brien, Susan; Byrd, John C.; Hillmen, Peter; Brown, Jennifer R.; Dyer, Martin J. S.; Mato, Anthony R.; Keating, Michael J.; Jaglowski, Samantha; Clow, Fong; Rezvani, Andrew R.; Styles, Lori; Coutre, Steven E.

2016-01-01

Ibrutinib, a potent and irreversible small-molecule inhibitor of both Bruton’s tyrosine kinase and interleukin-2 inducible kinase (ITK), has been used to treat relapsed/refractory chronic lymphocytic leukemia (CLL) with prolongation of progression-free and overall survival. Here, we present 27 patients with relapsed CLL following allogeneic hematopoietic cell transplant (HCT) who subsequently received ibrutinib salvage therapy. Sixteen of these patients were part of multi-institutional clinical trials and achieved an overall response rate of 87.5%. An additional 11 patients were treated at Stanford University following US Food and Drug Administration approval of ibrutinib; 7 (64%) achieved a complete response, and 3 (27%) achieved a partial response. Of the 9 patients treated at Stanford who had mixed chimerism–associated CLL relapse, 4 (44%) converted to full donor chimerism following ibrutinib initiation, in association with disease response. Four of 11 (36%) patients evaluated by ClonoSeq achieved minimal residual disease negativity with CLL <1/10 000 white blood cells, which persisted even after ibrutinib was discontinued, in 1 case even after 26 months. None of the 27 patients developed graft-versus-host-disease (GVHD) following ibrutinib initiation. We postulate that ibrutinib augments the graft-versus-leukemia (GVL) benefit through a T-cell–mediated effect, most likely due to ITK inhibition. To investigate the immune modulatory effects of ibrutinib, we completed comprehensive immune phenotype characterization of peripheral B and T cells from treated patients. Our results show that ibrutinib selectively targets pre–germinal B cells and depletes Th2 helper cells. Furthermore, these effects persisted after drug discontinuation. In total, our results provide evidence that ibrutinib effectively augments GVL without causing GVHD. PMID:27802969

p38 Mitogen-Activated Protein Kinase/Signal Transducer and Activator of Transcription-3 Pathway Signaling Regulates Expression of Inhibitory Molecules in T Cells Activated by HIV-1–Exposed Dendritic Cells

PubMed Central

Che, Karlhans Fru; Shankar, Esaki Muthu; Muthu, Sundaram; Zandi, Sasan; Sigvardsson, Mikael; Hinkula, Jorma; Messmer, Davorka; Larsson, Marie

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) infection enhances the expression of inhibitory molecules on T cells, leading to T-cell impairment. The signaling pathways underlying the regulation of inhibitory molecules and subsequent onset of T-cell impairment remain elusive. We showed that both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells (DCs) upregulated cytotoxic T-lymphocyte antigen (CTLA-4), tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), lymphocyte-activation gene-3 (LAG3), T-cell immunoglobulin mucin-3 (TIM-3), CD160 and certain suppression-associated transcription factors, such as B-lymphocyte induced maturation protein-1 (BLIMP-1), deltex homolog 1 protein (DTX1) and forkhead box P3 (FOXP3), leading to T-cell suppression. This induction was regulated by p38 mitogen-activated protein kinase/signal transducer and activator of transcription-3 (P38MAPK/STAT3) pathways, because their blockade significantly abrogated expression of all the inhibitory molecules studied and a subsequent recovery in T-cell proliferation. Neither interleukin-6 (IL-6) nor IL-10 nor growth factors known to activate STAT3 signaling events were responsible for STAT3 activation. Involvement of the P38MAPK/STAT3 pathways was evident because these proteins had a higher level of phosphorylation in the HIV-1–primed cells. Furthermore, blockade of viral CD4 binding and fusion significantly reduced the negative effects DCs imposed on primed T cells. In conclusion, HIV-1 interaction with DCs modulated their functionality, causing them to trigger the activation of the P38MAPK/STAT3 pathway in T cells, which was responsible for the upregulation of inhibitory molecules. PMID:22777388

Cyclin-dependent kinase 5-mediated phosphorylation of CHIP promotes the tAIF-dependent death pathway in rotenone-treated cortical neurons.

PubMed

Kim, Chiho; Lee, Juhyung; Ko, Yeon Uk; Oh, Young J

2018-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. Its dysregulation has been implicated in various neurodegenerative diseases. We previously reported that phosphorylation of the C-terminus of the Hsc70-interacting protein (CHIP) by Cdk5 promotes truncated apoptosis-inducing factor (tAIF)-mediated neuronal death induced by oxidative stress. Here, we determined whether this Cdk5-dependent cell death signaling pathway is present in experimental models of Parkinson's disease. First, we showed that rotenone activates Cdk5 in primary cultures of cortical neurons and causes tAIF-dependent neuronal cell death. This event was attenuated by negative regulation of endogenous Cdk5 activity by the pharmacological Cdk5 inhibitor, roscovitine, or by lentiviral knockdown of Cdk5. Cdk5 phosphorylates CHIP at Ser20 in rotenone-treated neurons. Consequently, overexpression of CHIP S20A , but not CHIP WT , attenuates tAIF-induced cell death in rotenone-treated cortical neurons. Taken together, these results indicate that phosphorylation of CHIP at Ser20 by Cdk5 activation inhibits CHIP-mediated tAIF degradation, thereby contributing to tAIF-induced neuronal cell death following rotenone treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters.

PubMed

Maryu, Gembu; Miura, Haruko; Uda, Youichi; Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

2018-04-25

Protein kinases play pivotal roles in intracellular signal transduction, and dysregulation of kinases leads to pathological results such as malignant tumors. Kinase activity has hitherto been measured by biochemical methods such as in vitro phosphorylation assay and western blotting. However, these methods are less useful to explore spatial and temporal changes in kinase activity and its cell-to-cell variation. Recent advances in fluorescent proteins and live-cell imaging techniques enable us to visualize kinase activity in living cells with high spatial and temporal resolutions. Several genetically encoded kinase activity reporters, which are based on the modes of action of kinase activation and phosphorylation, are currently available. These reporters are classified into single-fluorophore kinase activity reporters and Förster (or fluorescence) resonance energy transfer (FRET)-based kinase activity reporters. Here, we introduce the principles of genetically encoded kinase activity reporters, and discuss the advantages and disadvantages of these reporters.Key words: kinase, FRET, phosphorylation, KTR.

Distinct Effects of Saracatinib on Memory CD8+ T-cell Differentiation

PubMed Central

Takai, Shinji; Sabzevari, Helen; Farsaci, Benedetto; Schlom, Jeffrey; Greiner, John W.

2012-01-01

Immunologic memory involving CD8+ T-cells is a hallmark of an adaptive antigen-specific immune response and comprises a critical component of protective immunity. Designing approaches that enhance long-term T-cell memory would, for the most part, fortify vaccines and enhance host protection against infectious diseases and, perhaps, cancer immunotherapy. A better understanding of the cellular programs involved in the antigen-specific T-cell response has led to new approaches that target the magnitude and quality of the memory T-cell response. Here we show that T-cells from T-cell receptor transgenic mice for the nucleoprotein of influenza virus NP68 exhibit the distinct phases priming, expansion, contraction, memory - of an antigen-specific T-cell response when exposed in vitro to the cognate peptide. Saracatinib, a specific inhibitor of Src family kinases, administered at low doses during the expansion or contraction phases, increased CD62Lhigh/CD44high central memory CD8+ T-cells and IFN-γ production, while suppressing immunity when added during the priming phase. These effects by saracatinib were not accompanied by the expected decline of Src family kinases, but were accompanied by Akt-mTOR suppression and/or mediated via another pathway. Increased central memory cells by saracatinib were recapitulated in mice using a poxvirus-based influenza vaccine, thus underscoring the importance of dose and timing of the inhibitor in the context of memory T-cell differentiation. Finally, vaccine plus saracatinib treatment showed better protection against tumor challenge. The immune-potentiating effects on CD8+ T-cells by a low dose of saracatinib might afford better protection from pathogen or cancer when combined with vaccine. PMID:22450814

Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

PubMed

Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

2015-12-01

Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

PubMed Central

Datta, Antara; Silverman, Lee; Phipps, Andrew J; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D

2007-01-01

Background Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation. Results Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes. Conclusion Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival. PMID:17634129

Regulation of T cell homeostasis by JAKs and STATs.

PubMed

Ross, Jeremy A; Nagy, Zsuzsanna S; Cheng, Hanyin; Stepkowski, Stanislaw M; Kirken, Robert A

2007-01-01

Regulation of T cell homeostasis is critical for maintaining normal immune function. An imbalance in T cell proliferation can result in disorders ranging from cancer and autoimmunity to immunodeficiencies. Full activation of T cells requires three sequential signals, where signal 3, which is delivered by multiple cytokines, regulates proliferation, differentiation, and survival/death. Signaling from cytokines through their receptors is primarily delivered by two molecular families, namely Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs). Invaluable knowledge about JAKs and STATs has arisen from studies of mice made genetically deficient in these molecules, analyses of tumor models, and studies of expression patterns by proteomics/genomics, which all have begun to define the role of JAKs and STATs in survival versus apoptosis. These findings also have suggested ways in which JAKs and STATs may be manipulated for therapeutic intervention in lymphoid-derived diseases. This review seeks to focus on the role of JAK tyrosine kinases and STAT transcription factors in mediating the lymphocyte life cycle and how they might be manipulated for therapeutic applications.

Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.

PubMed

Amador, Erick; López-Pacheco, Karla; Morales, Nataly; Coria, Roberto; López-Villaseñor, Imelda

2017-04-01

Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

BRAF and MEK Inhibitors Influence the Function of Reprogrammed T Cells: Consequences for Adoptive T-Cell Therapy.

PubMed

Dörrie, Jan; Babalija, Lek; Hoyer, Stefanie; Gerer, Kerstin F; Schuler, Gerold; Heinzerling, Lucie; Schaft, Niels

2018-01-18

BRAF and MEK inhibitors (BRAFi/MEKi), the standard treatment for patients with BRAF V600 mutated melanoma, are currently explored in combination with various immunotherapies, notably checkpoint inhibitors and adoptive transfer of receptor-transfected T cells. Since two BRAFi/MEKi combinations with similar efficacy are approved, potential differences in their effects on immune cells would enable a rational choice for triple therapies. Therefore, we characterized the influence of the clinically approved BRAFi/MEKi combinations dabrafenib (Dabra) and trametinib (Tram) vs. vemurafenib (Vem) and cobimetinib (Cobi) on the activation and functionality of chimeric antigen receptor (CAR)-transfected T cells. We co-cultured CAR-transfected CD8⁺ T cells and target cells with clinically relevant concentrations of the inhibitors and determined the antigen-induced cytokine secretion. All BRAFi/MEKi reduced this release as single agents, with Dabra having the mildest inhibitory effect, and Dabra + Tram having a clearly milder inhibitory effect than Vem + Cobi. A similar picture was observed for the upregulation of the activation markers CD25 and CD69 on CAR-transfected T cells after antigen-specific stimulation. Most importantly, the cytolytic capacity of the CAR-T cells was significantly inhibited by Cobi and Vem + Cobi, whereas the other kinase inhibitors showed no effect. Therefore, the combination Dabra + Tram would be more suitable for combining with T-cell-based immunotherapy than Vem + Cobi.

Barium promotes anchorage-independent growth and invasion of human HaCaT keratinocytes via activation of c-SRC kinase.

PubMed

Thang, Nguyen Dinh; Yajima, Ichiro; Kumasaka, Mayuko Y; Ohnuma, Shoko; Yanagishita, Takeshi; Hayashi, Rumiko; Shekhar, Hossain U; Watanabe, Daisuke; Kato, Masashi

2011-01-01

Explosive increases in skin cancers have been reported in more than 36 million patients with arsenicosis caused by drinking arsenic-polluted well water. This study and previous studies showed high levels of barium as well as arsenic in the well water. However, there have been no reports showing a correlation between barium and cancer. In this study, we examined whether barium (BaCl(2)) may independently have cancer-related effects on human precancerous keratinocytes (HaCaT). Barium (5-50 µM) biologically promoted anchorage-independent growth and invasion of HaCaT cells in vitro. Barium (5 µM) biochemically enhanced activities of c-SRC, FAK, ERK and MT1-MMP molecules, which regulate anchorage-independent growth and/or invasion. A SRC kinase specific inhibitor, protein phosphatase 2 (PP2), blocked barium-mediated promotion of anchorage-independent growth and invasion with decreased c-SRC kinase activity. Barium (2.5-5 µM) also promoted anchorage-independent growth and invasion of fibroblasts (NIH3T3) and immortalized nontumorigenic melanocytes (melan-a), but not transformed cutaneous squamous cell carcinoma (HSC5 and A431) and malignant melanoma (Mel-ret) cells, with activation of c-SRC kinase. Taken together, our biological and biochemical findings newly suggest that the levels of barium shown in drinking well water independently has the cancer-promoting effects on precancerous keratinocytes, fibroblast and melanocytes in vitro.

PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

PubMed Central

Li, Qiongshu; Liu, Muyun; Wu, Man; Zhou, Xin; Wang, Shaobin; Hu, Yuan; Wang, Youfu; He, Yixin; Zeng, Xiaoping; Chen, Junhui; Liu, Qubo; Xiao, Dong; Hu, Xiang; Liu, Weibin

2018-01-01

Placenta-specific 1 (PLAC1), a novel cancer-testis antigen (CTA), is expressed in a number of different human malignancies. It is frequently produced in breast cancer, serving a function in tumorigenesis. Adoptive immunotherapy using T cell receptor (TCR)-engineered T cells against CTA mediates objective tumor regression; however, to the best of our knowledge, targeting PLAC1 using engineered T cells has not yet been attempted. In the present study, the cDNAs encoding TCRα- and β-chains specific for human leukocyte antigen (HLA)-A*0201-restricted PLAC1 were cloned from a cytotoxic T-lymphocyte, generated by in vitro by the stimulation of CD8+ T cells using autologous HLA-A2+ dendritic cells loaded with a PLAC1-specific peptide (p28-36, VLCSIDWFM). The TCRα/β-chains were linked by a 2A peptide linker (TCRα-Thosea asigna virus-TCRβ), and the constructs were cloned into the lentiviral vector, followed by transduction into human cytotoxic (CD8+) T cells. The efficiency of transduction was up to 25.16%, as detected by PLAC1 multimers. TCR-transduced CD8+ T cells, co-cultured with human non-metastatic breast cancer MCF-7 cells (PLAC1+, HLA-A2+) and triple-negative breast cancer MDAMB-231 cells (PLAC1+, HLA-A2+), produced interferon γ and tumor necrosis factor α, suggesting TCR activation. Furthermore, the PLAC1 TCR-transduced CD8+ T cells efficiently and specifically identified and annihilated the HLA-A2+/PLAC1+ breast cancer cell lines in a lactate dehydrogenase activity assay. Western blot analysis demonstrated that TCR transduction stimulated the production of mitogen-activated protein kinase signaling molecules, extracellular signal-regulated kinases 1/2 and nuclear factor-κB, through phosphoinositide 3-kinase γ-mediated phosphorylation of protein kinase B in CD8+ T cells. Xenograft mouse assays revealed that PLAC1 TCR-transduced CD8+T cells significantly delayed the tumor progression in mice-bearing breast cancer compared with normal saline or negative

Ibrutinib efficacy and tolerability in patients with relapsed chronic lymphocytic leukemia following allogeneic HCT.

PubMed

Ryan, Christine E; Sahaf, Bita; Logan, Aaron C; O'Brien, Susan; Byrd, John C; Hillmen, Peter; Brown, Jennifer R; Dyer, Martin J S; Mato, Anthony R; Keating, Michael J; Jaglowski, Samantha; Clow, Fong; Rezvani, Andrew R; Styles, Lori; Coutre, Steven E; Miklos, David B

2016-12-22

Ibrutinib, a potent and irreversible small-molecule inhibitor of both Bruton's tyrosine kinase and interleukin-2 inducible kinase (ITK), has been used to treat relapsed/refractory chronic lymphocytic leukemia (CLL) with prolongation of progression-free and overall survival. Here, we present 27 patients with relapsed CLL following allogeneic hematopoietic cell transplant (HCT) who subsequently received ibrutinib salvage therapy. Sixteen of these patients were part of multi-institutional clinical trials and achieved an overall response rate of 87.5%. An additional 11 patients were treated at Stanford University following US Food and Drug Administration approval of ibrutinib; 7 (64%) achieved a complete response, and 3 (27%) achieved a partial response. Of the 9 patients treated at Stanford who had mixed chimerism-associated CLL relapse, 4 (44%) converted to full donor chimerism following ibrutinib initiation, in association with disease response. Four of 11 (36%) patients evaluated by ClonoSeq achieved minimal residual disease negativity with CLL <1/10 000 white blood cells, which persisted even after ibrutinib was discontinued, in 1 case even after 26 months. None of the 27 patients developed graft-versus-host-disease (GVHD) following ibrutinib initiation. We postulate that ibrutinib augments the graft-versus-leukemia (GVL) benefit through a T-cell-mediated effect, most likely due to ITK inhibition. To investigate the immune modulatory effects of ibrutinib, we completed comprehensive immune phenotype characterization of peripheral B and T cells from treated patients. Our results show that ibrutinib selectively targets pre-germinal B cells and depletes Th2 helper cells. Furthermore, these effects persisted after drug discontinuation. In total, our results provide evidence that ibrutinib effectively augments GVL without causing GVHD. © 2016 by The American Society of Hematology.

Expression of activating natural killer-cell receptors is a hallmark of the innate-like T-cell neoplasm in peripheral T-cell lymphomas.

PubMed

Uemura, Yu; Isobe, Yasushi; Uchida, Akiko; Asano, Junko; Nishio, Yuji; Sakai, Hirotaka; Hoshikawa, Masahiro; Takagi, Masayuki; Nakamura, Naoya; Miura, Ikuo

2018-04-01

Peripheral T- or natural killer (NK)-cell lymphomas are rare and difficult-to-recognize diseases. It remains arduous to distinguish between NK cell- and cytotoxic T-lymphocyte-derived lymphomas through routine histological evaluation. To clarify the cells of origin, we focused on NK-cell receptors and examined the expression using immunohistochemistry in 22 cases with T- and NK-cell neoplasms comprising angioimmunoblastic T-cell lymphoma, anaplastic lymphoma kinase (ALK)-positive and -negative anaplastic large-cell lymphomas, extranodal NK/T-cell lymphoma, nasal type, monomorphic epitheliotropic intestinal T-cell lymphoma, aggressive NK-cell leukemia, and other peripheral T-cell lymphomas. Inhibitory receptor leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) was detected in 14 (64%) cases, whereas activating receptors DNAM1, NKp46, and NKG2D were expressed in 7 (32%), 9 (41%), and 5 (23%) cases, respectively. Although LILRB1 was detected regardless of the disease entity, the activating NK-cell receptors were expressed predominantly in TIA-1-positive neoplasms (DNAM1, 49%; NKp46, 69%; and NKG2D, 38%). In addition, NKp46 and NKG2D were detected only in NK-cell neoplasms and cytotoxic T-lymphocyte-derived lymphomas including monomorphic epitheliotropic intestinal T-cell lymphoma. One Epstein-Barr virus-harboring cytotoxic T-lymphocyte-derived lymphoma mimicking extranodal NK/T-cell lymphoma, nasal type lacked these NK-cell receptors, indicating different cell origin from NK and innate-like T cells. Furthermore, NKG2D expression showed a negative impact on survival among the 22 examined cases, which mainly received the standard chemotherapy regimen (log-rank test, P = .024). We propose that the presence of activating NK-cell receptors may provide new insights into understanding peripheral T-cell lymphomas and characterizing them as innate-like T-cell neoplasm. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on

Aurora A drives early signalling and vesicle dynamics during T-cell activation

PubMed Central

Blas-Rus, Noelia; Bustos-Morán, Eugenio; Pérez de Castro, Ignacio; de Cárcer, Guillermo; Borroto, Aldo; Camafeita, Emilio; Jorge, Inmaculada; Vázquez, Jesús; Alarcón, Balbino; Malumbres, Marcos; Martín-Cófreces, Noa B.; Sánchez-Madrid, Francisco

2016-01-01

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal. PMID:27091106

Plumbagin exerts an immunosuppressive effect on human T-cell acute lymphoblastic leukemia MOLT-4 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bae, Kyoung Jun; Lee, Yura; Kim, Soon Ae

2016-04-22

Of the hematological disorders typified by poor prognoses and survival rates, T-cell acute lymphoblastic leukemia (T-ALL) is one of the most commonly diagnosed. Despite the development of new therapeutic agents, the treatment options for this cancer remain limited. In this manuscript, we investigated the anti-proliferative effects of plumbagin, mediated by the activation of mitogen-activated protein kinase (MAPK) pathways, and inhibition of NF-κB signaling; the human T-ALL MOLT-4 cell line was used as our experimental system. Plumbagin is a natural, plant derived compound, which exerts an anti-proliferative activity against many types of human cancer. Our experiments confirm that plumbagin induces a caspase-dependentmore » apoptosis of MOLT-4 cells, with no significant cytotoxicity seen for normal peripheral blood mononuclear cells (PBMCs). Plumbagin also inhibited LPS-induced phosphorylation of p65, and the transcription of NF-κB target genes. Our results now show that plumbagin is a potent inhibitor of the NF-κB signaling pathway, and suppressor of T-ALL cell proliferation. - Highlights: • Plumbagin induces caspase-dependent apoptosis in T-ALL MOLT-4 cells. • Plumbagin activates phosphorylation of stress-activated protein kinase (SAPK) JNK and p38. • Plumbagin inhibits LPS-mediated NF-κB signaling cascade. • Plumbagin inhibits LPS-mediated transcriptional activity of pro-inflammatory cytokines.« less

Role of T Cells in Malnutrition and Obesity

PubMed Central

Gerriets, Valerie A.; MacIver, Nancie J.

2014-01-01

Nutritional status is critically important for immune cell function. While obesity is characterized by inflammation that promotes metabolic syndrome including cardiovascular disease and insulin resistance, malnutrition can result in immune cell defects and increased risk of mortality from infectious diseases. T cells play an important role in the immune adaptation to both obesity and malnutrition. T cells in obesity have been shown to have an early and critical role in inducing inflammation, accompanying the accumulation of inflammatory macrophages in obese adipose tissue, which are known to promote insulin resistance. How T cells are recruited to adipose tissue and activated in obesity is a topic of considerable interest. Conversely, T cell number is decreased in malnourished individuals, and T cells in the setting of malnutrition have decreased effector function and proliferative capacity. The adipokine leptin, which is secreted in proportion to adipocyte mass, may have a key role in mediating adipocyte-T cell interactions in both obesity and malnutrition, and has been shown to promote effector T cell function and metabolism while inhibiting regulatory T cell proliferation. Additionally, key molecular signals are involved in T cell metabolic adaptation during nutrient stress; among them, the metabolic regulator AMP kinase and the mammalian target of rapamycin have critical roles in regulating T cell number, function, and metabolism. In summary, understanding how T cell number and function are altered in obesity and malnutrition will lead to better understanding of and treatment for diseases where nutritional status determines clinical outcome. PMID:25157251

Polyunsaturated Fatty Acids Inhibit T Cell Signal Transduction by Modification of Detergent-insoluble Membrane Domains

PubMed Central

Stulnig, Thomas M.; Berger, Markus; Sigmund, Thomas; Raederstorff, Daniel; Stockinger, Hannes; Waldhäusl, Werner

1998-01-01

Polyunsaturated fatty acids (PUFAs) exert immunosuppressive effects, but the molecular alterations leading to T cell inhibition are not yet elucidated. Signal transduction seems to involve detergent-resistant membrane domains (DRMs) acting as functional rafts within the plasma membrane bilayer with Src family protein tyrosine kinases being attached to their cytoplasmic leaflet. Since DRMs include predominantly saturated fatty acyl moieties, we investigated whether PUFAs could affect T cell signaling by remodeling of DRMs. Jurkat T cells cultured in PUFA-supplemented medium showed a markedly diminished calcium response when stimulated via the transmembrane CD3 complex or glycosyl phosphatidylinositol (GPI)- anchored CD59. Immunofluorescence studies indicated that CD59 but not Src family protein tyrosine kinase Lck remained in a punctate pattern after PUFA enrichment. Analysis of DRMs revealed a marked displacement of Src family kinases (Lck, Fyn) from DRMs derived from PUFA-enriched T cells compared with controls, and the presence of Lck in DRMs strictly correlated with calcium signaling. In contrast, GPI-anchored proteins (CD59, CD48) and ganglioside GM1, both residing in the outer membrane leaflet, remained in the DRM fraction. In conclusion, PUFA enrichment selectively modifies the cytoplasmic layer of DRMs and this alteration could underlie the inhibition of T cell signal transduction by PUFAs. PMID:9813086

Tim-4 inhibition of T-cell activation and T helper type 17 differentiation requires both the immunoglobulin V and mucin domains and occurs via the mitogen-activated protein kinase pathway

PubMed Central

Cao, Wei; Ryan, Michelle; Buckley, Deirdre; O'Connor, Rosemary; Clarkson, Michael R

2011-01-01

Emerging experimental data suggest an important role for the T-cell immunoglobulin mucin 1 (Tim-1):Tim-4 pathway in autoimmune and alloimmune responses in vivo. Using a Tim-4 ectodomain human IgG Fc fusion protein we studied the role of Tim-4 in T-cell activation, signalling and differentiation responses in vitro. We demonstrate that Tim-4Fc can inhibit naive and pre-activated T-cell activation, proliferation and cytokine secretion via a Tim-1-independent pathway. Tim-4 contains immunoglobulin variable (IgV) and mucin domains; to identify which domain accounts for the inhibitory effect novel Tim-4 fusion proteins containing either the IgV or mucin domain were generated. We demonstrate that both IgV and mucin domains are required for the inhibitory effects and that they are mediated at least in part by inhibition of extracellular signal-regulated kinase pathway activity. Given the emerging interest in the role of the Tim family in T helper type 17 (Th17) cells, which play an important role in autoimmune disease and transplantation tolerance, our data show that Tim-4Fc can prevent polarization of CD4+ T cells to the Th17 phenotype. Collectively, our results highlight an inhibitory role for Tim-4Fc in vitro, which we propose is mediated by a receptor other than Tim-1. In addition, this study provides new insights into the role of Tim-4Fc in regulating Th17 immune responses and may open a new avenue for autoimmune therapy. PMID:21463297

Macroporous hydrogel micropillars for quantifying Met kinase activity in cancer cell lysates.

PubMed

Powers, Alicia D; Liu, Bi; Lee, Andrew G; Palecek, Sean P

2012-09-07

Overactive and overexpressed kinases have been implicated in the cause and progression of many cancers. Kinase inhibitors offer a targeted approach for treating cancers associated with increased or deregulated kinase activity. Often, however, cancer cells exhibit initial resistance to these inhibitors or evolve to develop resistance during treatment. Additionally, cancers of any one tissue type are typically heterogeneous in their oncogenesis mechanisms, and thus diagnosis of a particular type of cancer does not necessarily provide insight into what kinase therapies may be effective. For example, while some lung cancer cells that overexpress the epidermal growth factor receptor (EFGR) respond to treatment with EGFR kinase inhibitors, overexpression or hyperactivity of Met kinase correlates with resistance to EGFR kinase inhibitors. Here we describe a microfluidic-based assay for quantifying Met kinase activity in cancer cell lysates with the eventual goals of predicting cancer cell responsiveness to kinase inhibitors and monitoring development of resistance to these inhibitors. In this assay, we immobilized a phosphorylation substrate for Met kinase into macroporous hydrogel micropillars. We then exposed the micropillars to a cancer cell lysate and detected substrate phosphorylation using a fluorescently conjugated antibody. This assay is able to quantify Met kinase activity in whole cell lysate from as few as 150 cancer cells. It can also detect cells expressing overactive Met kinase in a background of up to 75% non-cancerous cells. Additionally, the assay can quantify kinase inhibition by the Met-specific kinase inhibitors SU11274 and PHA665752, suggesting predictive capability for cellular response to kinase inhibitors.

Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

PubMed

Chung, T; Huang, J S; Mukherjee, J J; Crilly, K S; Kiss, Z

2000-05-01

In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

PI3Kδ promotes CD4(+) T-cell interactions with antigen-presenting cells by increasing LFA-1 binding to ICAM-1.

PubMed

Garçon, Fabien; Okkenhaug, Klaus

2016-05-01

Activation of T lymphocytes by peptide/major histocompatibility complex on antigen-presenting cells (APCs) involves dynamic contacts between the two cells, during which T cells undergo marked morphological changes. These interactions are facilitated by integrins. Activation of the T cells increases the binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) expressed by T cells to intercellular adhesion molecule (ICAM)-1 and ICAM-2 expressed by APCs. The signalling pathways that control integrin affinities are incompletely defined. The phosphoinositide 3-kinases (PI3Ks) generate second-messenger signalling molecules that control cell growth, proliferation, differentiation and trafficking. Here we show that in T cells, PI3Kδ attenuates the activation of Rac1, but sustains the activation of Rap1. Consequently, PI3Kδ increases LFA-1-dependent adhesion to form stable conjugates with APCs. Increased Rap1 activity and LFA-1 adhesion were only in part mediated by the downstream kinase Akt, suggesting the involvement of additional phosphatidylinositol(3,4,5)P3-binding proteins. These results establish a link between PI3K activity, cytoskeletal changes and integrin binding and help explain the impaired T-cell-dependent immune responses in PI3Kδ-deficient mice.

ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vieira da Silva, Claudio; Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sao Paulo, Rua Botucatu, 862, 6o andar, 04023-062 Sao Paulo, SP; Alves da Silva, Erika

2009-01-16

Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RHmore » strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP{sub 2} and PIP{sub 3} to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.« less

The tax oncoprotein of human T-cell leukemia virus type 1 associates with and persistently activates IkappaB kinases containing IKKalpha and IKKbeta.

PubMed

Chu, Z L; DiDonato, J A; Hawiger, J; Ballard, D W

1998-06-26

The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV1) chronically activates transcription factor NF-kappaB by a mechanism involving degradation of IkappaBalpha, an NF-kappaB-associated cytoplasmic inhibitor. Tax-induced breakdown of IkappaBalpha requires phosphorylation of the inhibitor at Ser-32 and Ser-36, which is also a prerequisite for the transient activation of NF-kappaB in cytokine-treated T lymphocytes. However, it remained unclear how Tax interfaces with the cellular NF-kappaB/IkappaB signaling machinery to generate a chronic rather than a transient NF-kappaB response. We now demonstrate that Tax associates with cytokine-inducible IkappaB kinase (IKK) complexes containing catalytic subunits IKKalpha and IKKbeta, which mediate phosphorylation of IkappaBalpha at Ser-32 and Ser-36. Unlike their transiently activated counterparts in cytokine-treated cells, Tax-associated forms of IKK are constitutively active in either Tax transfectants or HTLV1-infected T lymphocytes. Moreover, point mutations in Tax that ablate its IKK-binding function also prevent Tax-mediated activation of IKK and NF-kappaB. Together, these findings suggest that the persistent activation of NF-kappaB in HTLV1-infected T-cells is mediated by a direct Tax/IKK coupling mechanism.

Differential regulation of p65 and c-Rel NF-kappaB transactivating activity by Cot, protein kinase C zeta and NIK protein kinases in CD3/CD28 activated T cells.

PubMed

Sánchez-Valdepeñas, Carmen; Punzón, Carmen; San-Antonio, Belén; Martin, Angel G; Fresno, Manuel

2007-03-01

It has been shown that phosphorylation of p65/RelA and c-Rel plays a role in the regulation of transcriptional activity of NF-kappaB independent on IkappaB degradation. In this study, we show that anti CD3/CD28 activation induces the transactivation activity of both p65/RelA and c-Rel in T cells using Gal4 dependent assays. Moreover, protein kinase C (PKC)zeta, Cot kinase and NF-kappaB-inducing kinase (NIK) seem to be involved in those processes in a different manner. Thus, transfection of dominant negative forms of Cot and PKCzeta inhibits CD3/CD28 induction of Gal4-p65 transactivation, whereas the kinase inactive versions of the 3 kinases inhibit induction of Gal4-c-Rel. Cot induction of Gal4-c-Rel transactivating activity seems to be mediated sequentially through PKCzeta and NIK activation, since dominant negative form of NIK blocks Cot and PKCzeta induction, whereas kinase inactive PKCzeta only blocks Cot activity. In contrast, the contribution of NIK to the transactivation function of p65/RelA seems to be negligible and more importantly NIK-KD did not inhibit induction by Cot and PKCzeta. Besides, the enhancing effect of Cot on Gal4-p65 was not decreased in mouse embryo fibroblasts from NIK deficient aly/aly mice in contrast with a greatest reduction on Gal4-c-Rel. By using Ser to Ala mutants in p65 and c-Rel transactivation domains, PKCzeta and NIK activities seem to be dependent of a restricted set of Ser in both proteins. In contrast, the enhancing effect of Cot seems to be less dependent of a particular set of Ser residues being partially abrogated by mutation of several Ser residues.

Regulation and function of mTOR signalling in T cell fate decision

PubMed Central

Chi, Hongbo

2012-01-01

The evolutionary conserved kinase mTOR couples cell growth and metabolism to environmental inputs in eukaryotes. T cells depend on mTOR signalling to integrate immune signals and metabolic cues for their proper maintenance and activation. Under steady-state conditions, mTOR is actively controlled by multiple inhibitory mechanisms, and this enforces normal T cell homeostasis. Antigen recognition by naïve CD4+ and CD8+ T cells triggers mTOR activation, which in turn programs their differentiation into functionally distinct lineages. This Review focuses on the signalling mechanisms of mTOR in T cell homeostatic and functional fates and therapeutic implications of targeting mTOR in T cells. PMID:22517423

Barium Promotes Anchorage-Independent Growth and Invasion of Human HaCaT Keratinocytes via Activation of c-SRC Kinase

PubMed Central

Thang, Nguyen Dinh; Yajima, Ichiro; Kumasaka, Mayuko Y.; Ohnuma, Shoko; Yanagishita, Takeshi; Hayashi, Rumiko; Shekhar, Hossain U.; Watanabe, Daisuke; Kato, Masashi

2011-01-01

Explosive increases in skin cancers have been reported in more than 36 million patients with arsenicosis caused by drinking arsenic-polluted well water. This study and previous studies showed high levels of barium as well as arsenic in the well water. However, there have been no reports showing a correlation between barium and cancer. In this study, we examined whether barium (BaCl2) may independently have cancer-related effects on human precancerous keratinocytes (HaCaT). Barium (5–50 µM) biologically promoted anchorage-independent growth and invasion of HaCaT cells in vitro. Barium (5 µM) biochemically enhanced activities of c-SRC, FAK, ERK and MT1-MMP molecules, which regulate anchorage-independent growth and/or invasion. A SRC kinase specific inhibitor, protein phosphatase 2 (PP2), blocked barium-mediated promotion of anchorage-independent growth and invasion with decreased c-SRC kinase activity. Barium (2.5–5 µM) also promoted anchorage-independent growth and invasion of fibroblasts (NIH3T3) and immortalized nontumorigenic melanocytes (melan-a), but not transformed cutaneous squamous cell carcinoma (HSC5 and A431) and malignant melanoma (Mel-ret) cells, with activation of c-SRC kinase. Taken together, our biological and biochemical findings newly suggest that the levels of barium shown in drinking well water independently has the cancer-promoting effects on precancerous keratinocytes, fibroblast and melanocytes in vitro. PMID:22022425

The tyrosine kinase inhibitor ZD6474 blocks proliferation of RET mutant medullary thyroid carcinoma cells.

PubMed

Vitagliano, Donata; De Falco, Valentina; Tamburrino, Anna; Coluzzi, Sabrina; Troncone, Giancarlo; Chiappetta, Gennaro; Ciardiello, Fortunato; Tortora, Giampaolo; Fagin, James A; Ryan, Anderson J; Carlomagno, Francesca; Santoro, Massimo

2011-02-01

Oncogenic conversion of the RET tyrosine kinase is a frequent feature of medullary thyroid carcinoma (MTC). ZD6474 (vandetanib) is an ATP-competitive inhibitor of RET, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptors kinases. In this study, we have studied ZD6474 mechanism of action in TT and MZ-CRC-1 human MTC cell lines, carrying cysteine 634 to tryptophan (C634W) and methionine 918 to threonine (M918T) RET mutation respectively. ZD6474 blunted MTC cell proliferation and RET, Shc and p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation. Single receptor knockdown by RNA interference showed that MTC cells depended on RET for proliferation. Adoptive expression of the ZD6474-resistant V804M RET mutant rescued proliferation of TT cells under ZD6474 treatment, showing that RET is a key ZD6474 target in these MTC cells. Upon RET inhibition, adoptive stimulation of EGFR partially rescued TT cell proliferation, MAPK signaling, and expression of cell-cycle-related genes. This suggests that simultaneous inhibition of RET and EGFR by ZD6474 may overcome the risk of MTC cells to escape from RET blockade through compensatory over-activation of EGFR.

Third-generation epidermal growth factor receptor-tyrosine kinase inhibitors in T790M-positive non-small cell lung cancer: review on emerged mechanisms of resistance

PubMed Central

Minari, Roberta; Bordi, Paola

2016-01-01

Osimertinib, third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), has been approved in the US and EU for the treatment of EGFR mutant T790M-positive non-small cell lung cancer (NSCLC) patients resistant to first- or second-generation EGFR-TKIs, such as gefitinib, erlotinib and afatinib. Although exciting survival data and response rates have been registered in patients treated with this and other third-generation EGFR-TKIs, unfortunately acquired resistance still occurs after approximately 10 months. Mechanisms determining progression of disease are heterogeneous and not fully understood. EGFR-dependent resistance mechanisms (such as new EGFR mutations), bypass pathway activation [as erb-b2 receptor tyrosine kinase 2 (HER2) or MET amplification] and histological transformation [in small cell lung cancer (SCLC)] have been reported, similarly to previous generation TKIs. Here, we review principle mechanisms of innate and acquired resistance described in literature both in clinical and preclinical settings during NSCLC treatment with third-generation EGFR-TKIs. PMID:28149764

Alteration of the gene expression profile of T-cell receptor αβ-modified T-cells with diffuse large B-cell lymphoma specificity.

PubMed

Zha, Xianfeng; Yin, Qingsong; Tan, Huo; Wang, Chunyan; Chen, Shaohua; Yang, Lijian; Li, Bo; Wu, Xiuli; Li, Yangqiu

2013-05-01

Antigen-specific, T-cell receptor (TCR)-modified cytotoxic T lymphocytes (CTLs) that target tumors are an attractive strategy for specific adoptive immunotherapy. Little is known about whether there are any alterations in the gene expression profile after TCR gene transduction in T cells. We constructed TCR gene-redirected CTLs with specificity for diffuse large B-cell lymphoma (DLBCL)-associated antigens to elucidate the gene expression profiles of TCR gene-redirected T-cells, and we further analyzed the gene expression profile pattern of these redirected T-cells by Affymetrix microarrays. The resulting data were analyzed using Bioconductor software, a two-fold cut-off expression change was applied together with anti-correlation of the profile ratios to render the microarray analysis set. The fold change of all genes was calculated by comparing the three TCR gene-modified T-cells and a negative control counterpart. The gene pathways were analyzed using Bioconductor and Kyoto Encyclopedia of Genes and Genomes. Identical genes whose fold change was greater than or equal to 2.0 in all three TCR gene-redirected T-cell groups in comparison with the negative control were identified as the differentially expressed genes. The differentially expressed genes were comprised of 33 up-regulated genes and 1 down-regulated gene including JUNB, FOS, TNF, INF-γ, DUSP2, IL-1B, CXCL1, CXCL2, CXCL9, CCL2, CCL4, and CCL8. These genes are mainly involved in the TCR signaling, mitogen-activated protein kinase signaling, and cytokine-cytokine receptor interaction pathways. In conclusion, we characterized the gene expression profile of DLBCL-specific TCR gene-redirected T-cells. The changes corresponded to an up-regulation in the differentiation and proliferation of the T-cells. These data may help to explain some of the characteristics of the redirected T-cells.

SENIEUR status of the originating cell donor negates certain 'anti-immunosenescence' effects of ebselen and N-acetyl cysteine in human T cell clone cultures.

PubMed

Marthandan, Shiva; Freeburn, Robin; Steinbrecht, Susanne; Pawelec, Graham; Barnett, Yvonne

2014-01-01

Damage to T cells of the immune system by reactive oxygen species may result in altered cell function or cell death and thereby potentially impact upon the efficacy of a subsequent immune response. Here, we assess the impact of the antioxidants Ebselen and N-acetyl cysteine on a range of biological markers in human T cells derived from a SENIEUR status donor. In addition, the impact of these antioxidants on different MAP kinase pathways in T cells from donors of different ages was also examined. T cell clones were derived from healthy 26, 45 and SENIEUR status 80 year old people and the impact of titrated concentrations of Ebselen or N-acetyl cysteine on their proliferation and in vitro lifespan, GSH:GSSG ratio as well as levels of oxidative DNA damage and on MAP kinase signaling pathways was examined. In this investigation neither Ebselen nor N-acetyl cysteine supplementation had any impact on the biological endpoints examined in the T cells derived from the SENIEUR status 80 year old donor. This is in contrast to the anti-immunosenescent effects of these antioxidants on T cells from donors of 26 or 45 years of age. The analysis of MAP kinases showed that pro-apoptotic pathways become activated in T cells with increasing in vitro age and that Ebselen or N-acetyl cysteine could decrease activation (phosphorylation) in T cells from 26 or 45 year old donors, but not from the SENIEUR status 80 year old donor. The results of this investigation demonstrate that the biological phenotype of SENIEUR status derived human T cells negates the anti-immunosenescence effects of Ebselen and also N-acetyl cysteine. The results highlight the importance of pre-antioxidant intervention evaluation to determine risk-benefit.

Autoreactive T Cells and Chronic Fungal Infection Drive Esophageal Carcinogenesis

PubMed Central

Zhu, Feng; Willette-Brown, Jami; Song, Na-Young; Lomada, Dakshayani; Song, Yongmei; Xue, Liyan; Gray, Zane; Zhao, Zitong; Davis, Sean R.; Sun, Zhonghe; Zhang, Peilin; Wu, Xiaolin; Zhan, Qimin; Richie, Ellen R.; Hu, Yinling

2018-01-01

SUMMARY Humans with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a T cell–driven autoimmune disease caused by impaired central tolerance, are susceptible to developing chronic fungal infection and esophageal squamous cell carcinoma (ESCC). However, the relationship between autoreactive T cells and chronic fungal infection in ESCC development remains unclear. We find that kinase-dead Ikkα knockin mice develop phenotypes reminiscent of APECED, including impaired central tolerance, autoreactive T cells, chronic fungal infection, and ESCCs expressing specific human ESCC markers. Using this model, we investigated the potential link between ESCC and fungal infection. Autoreactive CD4 T cells permit fungal infection and incite tissue injury and inflammation. Antifungal treatment or depletion of autoreactive CD4 T cells rescues, whereas oral fungal administration promotes, ESCC development. Inhibition of inflammation or EGFR activity decreases fungal burden. Importantly, fungal infection is highly associated with ESCCs in non-autoimmune human patients. Therefore, autoreactive T cells and chronic fungal infection, fostered by inflammation and epithelial injury, promote ESCC development. PMID:28407484

Helicobacter pylori induces cell migration and invasion through casein kinase 2 in gastric epithelial cells.

PubMed

Lee, Yeo Song; Lee, Do Yeon; Yu, Da Yeon; Kim, Shin; Lee, Yong Chan

2014-12-01

Chronic infection with Helicobacter pylori (H. pylori) is causally linked with gastric carcinogenesis. Virulent H. pylori strains deliver bacterial CagA into gastric epithelial cells. Induction of high motility and an elongated phenotype is considered to be CagA-dependent process. Casein kinase 2 plays a critical role in carcinogenesis through signaling pathways related to the epithelial mesenchymal transition. This study was aimed to investigate the effect of H. pylori infection on the casein kinase 2-mediated migration and invasion in gastric epithelial cells. AGS or MKN28 cells as human gastric epithelial cells and H. pylori strains Hp60190 (ATCC 49503, CagA(+)) and Hp8822 (CagA(-)) were used. Cells were infected with H. pylori at multiplicity of infection of 100 : 1 for various times. We measured in vitro kinase assay to examine casein kinase 2 activity and performed immunofluorescent staining to observe E-cadherin complex. We also examined β-catenin transactivation through promoter assay and MMP7 expression by real-time PCR and ELISA. H. pylori upregulates casein kinase 2 activity and inhibition of casein kinase 2 in H. pylori-infected cells profoundly suppressed cell invasiveness and motility. We confirmed that casein kinase 2 mediates membranous α-catenin depletion through dissociation of the α-/β-catenin complex in H. pylori-infected cells. We also found that H. pylori induces β-catenin nuclear translocation and increases MMP7 expressions mediated through casein kinase 2. We show for the first time that CagA(+) H. pylori upregulates cellular invasiveness and motility through casein kinase 2. The demonstration of a mechanistic interplay between H. pylori and casein kinase 2 provides important insights into the role of CagA(+) H. pylori in the gastric cancer invasion and metastasis. © 2014 John Wiley & Sons Ltd.

Function of the nucleotide exchange activity of vav1 in T cell development and activation.

PubMed

Saveliev, Alexander; Vanes, Lesley; Ksionda, Olga; Rapley, Jonathan; Smerdon, Stephen J; Rittinger, Katrin; Tybulewicz, Victor L J

2009-12-15

The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal-regulated kinase and protein kinase D1, and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions.

Function of the Nucleotide Exchange Activity of Vav1 in T cell Development and Activation*

PubMed Central

Saveliev, Alexander; Vanes, Lesley; Ksionda, Olga; Rapley, Jonathan; Smerdon, Stephen J.; Rittinger, Katrin; Tybulewicz, Victor L. J.

2012-01-01

The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal–regulated kinase (ERK) and protein kinase D1 (PKD1), and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions. PMID:20009105

SLP-76-ADAP adaptor module regulates LFA-1 mediated costimulation and T cell motility.

PubMed

Wang, Hongyan; Wei, Bin; Bismuth, Georges; Rudd, Christopher E

2009-07-28

Although adaptor ADAP (FYB) and its binding to SLP-76 has been implicated in TcR-induced "inside-out" signaling for LFA-1 activation in T cells, little is known regarding its role in LFA-1-mediated "outside-in" signaling. In this study, we demonstrate that ADAP and SLP-76-ADAP binding are coupled to LFA-1 costimulation of IL-2 production, F-actin clustering, cell polarization, and T cell motility. LFA-1 enhancement of anti-CD3-induced IL-2 production was completely dependent on SLP-76-ADAP binding. Further, anti-CD3 was found to require CD11a ligation by antibody or ICAM1 to cause T cell polarization. ADAP augmented this polarization induced by anti-CD3/CD11a, but not by anti-CD3 alone. ADAP expression with LFA-1 ligation alone was sufficient to polarize T cells directly and to increase T cell motility whereas the loss of ADAP in ADAP-/- primary T cells reduced motility. A mutant lacking SLP-76-binding sites (M12) blocked LFA-1 costimulation of IL-2 production, polarization, and motility. LFA-1-ADAP polarization was also dependent on src kinases, Rho GTPases, phospholipase C, and phosphoinositol 3-kinase. Our findings provide evidence of an obligatory role for the SLP-76-ADAP module in LFA-1-mediated costimulation in T cells.

Sezary syndrome cells unlike normal circulating T lymphocytes fail to migrate following engagement of NT1 receptor.

PubMed

Magazin, Marilyn; Poszepczynska-Guigné, Ewa; Bagot, Martine; Boumsell, Laurence; Pruvost, Christelle; Chalon, Pascale; Culouscou, Jean-Michel; Ferrara, Pascual; Bensussan, Armand

2004-01-01

Circulating malignant Sezary cells are a clonal proliferation of CD4+CD45RO+ T lymphocytes primarily involving the skin. To study the biology of these malignant T lymphocytes, we tested their ability to migrate in chemotaxis assays. Previously, we had shown that the neuropeptide neurotensin (NT) binds to freshly isolated Sezary malignant cells and induces through NT1 receptors the cell migration of the cutaneous T cell lymphoma cell line Cou-L. Here, we report that peripheral blood Sezary cells as well as the Sezary cell line Pno fail to migrate in response to neurotensin although they are capable of migrating to the chemokine stromal-cell-derived factor 1 alpha. This is in contrast with normal circulating CD4+ or CD8+ lymphocytes, which respond to both types of chemoattractants except after ex vivo short-time anti-CD3 monoclonal antibody activation, which abrogates the neurotensin-induced lymphocyte migration. Furthermore, we demonstrate that neurotensin-responsive T lymphocytes express the functional NT1 receptor responsible for chemotaxis. In these cells, but not in Sezary cells, neurotensin induces recruitment of phosphatidylinositol-3 kinase, and redistribution of phosphorylated cytoplasmic tyrosine kinase focal adhesion kinase and filamentous actin. Taken together, these results, which show functional distinctions between normal circulating lymphocytes and Sezary syndrome cells, contribute to further understanding of the physiopathology of these atypical cells.

RON kinase isoforms demonstrate variable cell motility in normal cells.

PubMed

Greenbaum, Alissa; Rajput, Ashwani; Wan, Guanghua

2016-09-01

Aberrant RON (Recepteur d'Origine Nantais) tyrosine kinase activation causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis. Wildtype (wt) RON has been shown to play a role in metastasis of epithelial malignancies. It presents an important potential therapeutic target for colorectal, breast, gastric and pancreatic cancer. Little is known about functional differences amongst RON isoforms RON155, RON160 and RON165. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility. Cell lines with stable expression of wtRON were generated by inserting the coding region of RON in pTagRFP (tagged red fluorescence protein plasmid). The expression constructs of RON variants (RON155, RON160 and RON165) were generated by creating a mutagenesis-based wtRON-pTag RFP plasmid and stably transfected into HEK 293 cells. The wound closure scratch assay was used to investigate the effect on cell migratory capacity of wild type RON and its variants. RON transfected cells demonstrated increased cell motility compared to HEK293 control cells. RON165 cell motility was significantly increased compared to RON160 (mean percentage of wound covered 37.37% vs. 32.40%; p = 0.03). RON tyrosine kinase isoforms have variable cell motility. This may reflect a difference in the behavior of malignant epithelial cells and their capacity for metastasis.

Protein Kinases and Phosphatases in the Control of Cell Fate

PubMed Central

Bononi, Angela; Agnoletto, Chiara; De Marchi, Elena; Marchi, Saverio; Patergnani, Simone; Bonora, Massimo; Giorgi, Carlotta; Missiroli, Sonia; Poletti, Federica; Rimessi, Alessandro; Pinton, Paolo

2011-01-01

Protein phosphorylation controls many aspects of cell fate and is often deregulated in pathological conditions. Several recent findings have provided an intriguing insight into the spatial regulation of protein phosphorylation across different subcellular compartments and how this can be finely orchestrated by specific kinases and phosphatases. In this review, the focus will be placed on (i) the phosphoinositide 3-kinase (PI3K) pathway, specifically on the kinases Akt and mTOR and on the phosphatases PP2a and PTEN, and on (ii) the PKC family of serine/threonine kinases. We will look at general aspects of cell physiology controlled by these kinases and phosphatases, highlighting the signalling pathways that drive cell division, proliferation, and apoptosis. PMID:21904669

Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors.

PubMed

Cannon, C M; Pozniak, J; Scott, M C; Ito, D; Gorden, B H; Graef, A J; Modiano, J F

2015-03-01

We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half-maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152-induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi-agent protocols is warranted. © 2013 Blackwell Publishing Ltd.

On the role of phosphatidylinositol 3-kinase, protein kinase b/Akt, and glycogen synthase kinase-3β in photodynamic injury of crayfish neurons and glial cells.

PubMed

Komandirov, Maxim A; Knyazeva, Evgeniya A; Fedorenko, Yulia P; Rudkovskii, Mikhail V; Stetsurin, Denis A; Uzdensky, Anatoly B

2011-10-01

Photodynamic treatment that causes intense oxidative stress and cell death is currently used in neurooncology. However, along with tumor cells, it may damage healthy neurons and glia. To study the involvement of signaling processes in photodynamic injury or protection of neurons and glia, we used crayfish mechanoreceptor consisting of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens. Application of specific inhibitors showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glia but its components, Akt and glycogen synthase kinase-3β, independently and cell specifically regulated death of neurons and glial cells. According to these data, necrosis in this system was a controlled but not a non-regulated cell death mode. The obtained results may be used for the search of pharmacological agents selectively modulating death and survival of normal neurons and glial cells during photodynamic therapy of brain tumors.

Targets of B-cell antigen receptor signaling: the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase-3 signaling pathway and the Rap1 GTPase.

PubMed

Gold, M R; Ingham, R J; McLeod, S J; Christian, S L; Scheid, M P; Duronio, V; Santos, L; Matsuuchi, L

2000-08-01

In this review, we discuss the role of phosphatidylinositol 3-kinase (PI3K) and Rap 1 in B-cell receptor (BCR) signaling. PI3K produces lipids that recruit pleckstrin homology domain-containing proteins to the plasma membrane. Akt is a kinase that the BCR activates in this manner. Akt phosphorylates several transcription factors as well as proteins that regulate apoptosis and protein synthesis. Akt also regulates glycogen synthase kinase-3, a kinase whose substrates include the nuclear factor of activated T cells (NF-AT)cl and beta-catenin transcriptional activators. In addition to Akt, PI3K-derived lipids also regulate the activity and localization of other targets of BCR signaling. Thus, a key event in BCR signaling is the recruitment of PI3K to the plasma membrane where its substrates are located. This is mediated by binding of the Src homology (SH) 2 domains in PI3K to phosphotyrosine-containing sequences on membrane-associated docking proteins. The docking proteins that the BCR uses to recruit PI3K include CD19, Cbl, Gab1, and perhaps Gab2. We have shown that Gab1 colocalizes PI3K with SH2 domain-containing inositol phosphatase (SHIP) and SHP2, two enzymes that regulate PI3K-dependent signaling. In contrast to PI3K, little is known about the Rap1 GTPase. We showed that the BCR activates Rap1 via phospholipase C-dependent production of diacylglycerol. Since Rap1 is thought to regulate cell adhesion and cell polarity, it may be involved in B-cell migration.

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

PubMed

Roskoski, Robert

2005-11-11

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

PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway.

PubMed

Desai, S; Pillai, P; Win-Piazza, H; Acevedo-Duncan, M

2011-06-01

The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway. Published by Elsevier B.V.

K-RAS GTPase- and B-RAF kinase-mediated T-cell tolerance defects in rheumatoid arthritis.

PubMed

Singh, Karnail; Deshpande, Pratima; Li, Guangjin; Yu, Mingcan; Pryshchep, Sergey; Cavanagh, Mary; Weyand, Cornelia M; Goronzy, Jörg J

2012-06-19

Autoantibodies to common autoantigens and neoantigens, such as IgG Fc and citrullinated peptides, are immunological hallmarks of rheumatoid arthritis (RA). We examined whether a failure in maintaining tolerance is mediated by defects in T-cell receptor activation threshold settings. RA T cells responded to stimulation with significantly higher ERK phosphorylation (P < 0.001). Gene expression arrays of ERK pathway members suggested a higher expression of KRAS and BRAF, which was confirmed by quantitative PCR (P = 0.003), Western blot, and flow cytometry (P < 0.01). Partial silencing of KRAS and BRAF lowered activation-induced phosphorylated ERK levels (P < 0.01). In individual cells, levels of these signaling molecules correlated with ERK phosphorylation, attesting that their concentrations are functionally important. In confocal studies, B-RAF/K-RAS clustering was increased in RA T cells 2 min after T-cell receptor stimulation (P < 0.001). Overexpression of B-RAF and K-RAS in normal CD4 T cells amplified polyclonal T-cell proliferation and facilitated responses to citrullinated peptides. We propose that increased expression of B-RAF and K-RAS lowers T-cell activation thresholds in RA T cells, enabling responses to autoantigens.

PaASK1, a mitogen-activated protein kinase kinase kinase that controls cell degeneration and cell differentiation in Podospora anserina.

PubMed Central

Kicka, Sébastien; Silar, Philippe

2004-01-01

MAPKKK are kinases involved in cell signaling. In fungi, these kinases are known to regulate development, pathogenicity, and the sensing of external conditions. We show here that Podospora anserina strains mutated in PaASK1, a MAPKKK of the MEK family, are impaired in the development of crippled growth, a cell degeneration process caused by C, a nonconventional infectious element. They also display defects in mycelium pigmentation, differentiation of aerial hyphae, and making of fruiting bodies, three hallmarks of cell differentiation during stationary phase in P. anserina. Overexpression of PaASK1 results in exacerbation of crippled growth. PaASK1 is a large protein of 1832 amino acids with several domains, including a region rich in proline and a 60-amino-acid-long polyglutamine stretch. Deletion analysis reveals that the polyglutamine stretch is dispensable for PaASK1 activity, whereas the region that contains the prolines is essential but insufficient to promote full activity. We discuss a model based on the hysteresis of a signal transduction cascade to account for the role of PaASK1 in both cell degeneration and stationary-phase cell differentiation. PMID:15082544

PaASK1, a mitogen-activated protein kinase kinase kinase that controls cell degeneration and cell differentiation in Podospora anserina.

PubMed

Kicka, Sébastien; Silar, Philippe

2004-03-01

MAPKKK are kinases involved in cell signaling. In fungi, these kinases are known to regulate development, pathogenicity, and the sensing of external conditions. We show here that Podospora anserina strains mutated in PaASK1, a MAPKKK of the MEK family, are impaired in the development of crippled growth, a cell degeneration process caused by C, a nonconventional infectious element. They also display defects in mycelium pigmentation, differentiation of aerial hyphae, and making of fruiting bodies, three hallmarks of cell differentiation during stationary phase in P. anserina. Overexpression of PaASK1 results in exacerbation of crippled growth. PaASK1 is a large protein of 1832 amino acids with several domains, including a region rich in proline and a 60-amino-acid-long polyglutamine stretch. Deletion analysis reveals that the polyglutamine stretch is dispensable for PaASK1 activity, whereas the region that contains the prolines is essential but insufficient to promote full activity. We discuss a model based on the hysteresis of a signal transduction cascade to account for the role of PaASK1 in both cell degeneration and stationary-phase cell differentiation.

Hippo kinases maintain polarity during directional cell migration in Caenorhabditis elegans.

PubMed

Feng, Guoxin; Zhu, Zhiwen; Li, Wen-Jun; Lin, Qirong; Chai, Yongping; Dong, Meng-Qiu; Ou, Guangshuo

2017-02-01

Precise positioning of cells is crucial for metazoan development. Despite immense progress in the elucidation of the attractive cues of cell migration, the repulsive mechanisms that prevent the formation of secondary leading edges remain less investigated. Here, we demonstrate that Caenorhabditis elegans Hippo kinases promote cell migration along the anterior-posterior body axis via the inhibition of dorsal-ventral (DV) migration. Ectopic DV polarization was also demonstrated in gain-of-function mutant animals for C. elegans RhoG MIG-2. We identified serine 139 of MIG-2 as a novel conserved Hippo kinase phosphorylation site and demonstrated that purified Hippo kinases directly phosphorylate MIG-2 S139 Live imaging analysis of genome-edited animals indicates that MIG-2 S139 phosphorylation impedes actin assembly in migrating cells. Intriguingly, Hippo kinases are excluded from the leading edge in wild-type cells, while MIG-2 loss induces uniform distribution of Hippo kinases. We provide evidence that Hippo kinases inhibit RhoG activity locally and are in turn restricted to the cell body by RhoG-mediated polarization. Therefore, we propose that the Hippo-RhoG feedback regulation maintains cell polarity during directional cell motility. © 2016 The Authors.

Malignant transformation of CD4+ T lymphocytes mediated by oncogenic kinase NPM/ALK recapitulates IL-2-induced cell signaling and gene expression reprogramming

PubMed Central

Marzec, Michal; Halasa, Krzysztof; Liu, Xiaobin; Wang, Hong Y.; Cheng, Mangeng; Baldwin, Donald; Tobias, John W.; Schuster, Stephen J.; Woetmann, Anders; Zhang, Qian; Turner, Suzanne D.; Odum, Niels; Wasik, Mariusz A.

2013-01-01

Anaplastic lymphoma kinase (ALK) physiologically expressed only by nervous system cells displays remarkable capacity to transform CD4+ T lymphocytes and other types of non-neural cells. Here we report that activity of nucleophosphmin (NPM)/ALK chimeric protein, the dominant form of ALK expressed in T-cell lymphomas (ALK+TCL), closely resembles cell activation induced by interleukin 2 (IL-2), the key cytokine supporting growth and survival of normal CD4+ T lymphocytes. Direct comparison of gene expression by ALK+TCL cells treated with an ALK inhibitor and IL-2-dependent ALK-TCL cells stimulated with the cytokine revealed a very similar, albeit inverse, gene regulation pattern. Depending on the analysis method, up to 67% of the modulated genes could be defined as modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT and IL-2 signaling pathways topped the list of pathways identified as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes: CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4 was confirmed at the protein level. In both ALK+TCL and IL-2-stimulated ALK-TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly by the STAT5 and STAT3 transcription factors, while transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms the target CD4+ T lymphocytes, at least in part, by utilizing the pre-existing, IL-2-dependent signaling pathways. PMID:24218456

Protein Kinase D1 attenuates tumorigenesis in colon cancer by modulating β-catenin/T cell factor activity

PubMed Central

Sundram, Vasudha; Ganju, Aditya; Hughes, Joshua E.; Khan, Sheema; Chauhan, Subhash C.; Jaggi, Meena

2014-01-01

Over 80% of colon cancer development and progression is a result of the dysregulation of β-catenin signaling pathway. Herein, for the first time, we demonstrate that a serine-threonine kinase, Protein Kinase D1 (PKD1), modulates the functions of β-catenin to suppress colon cancer growth. Analysis of normal and colon cancer tissues reveals downregulation of PKD1 expression in advanced stages of colon cancer and its co-localization with β-catenin in the colon crypts. This PKD1 downregulation corresponds with the aberrant expression and nuclear localization of β-catenin. In-vitro investigation of the PKD1-β-catenin interaction in colon cancer cells reveal that PKD1 overexpression suppresses cell proliferation and clonogenic potential and enhances cell-cell aggregation. We demonstrate that PKD1 directly interacts with β-catenin and attenuates β-catenin transcriptional activity by decreasing nuclear β-catenin levels. Additionally, we show that inhibition of nuclear β-catenin transcriptional activity is predominantly influenced by nucleus targeted PKD1. This subcellular modulation of β-catenin results in enhanced membrane localization of β-catenin and thereby increases cell-cell adhesion. Studies in a xenograft mouse model indicate that PKD1 overexpression delayed tumor appearance, enhanced necrosis and lowered tumor hypoxia. Overall, our results demonstrate a putative tumor-suppressor function of PKD1 in colon tumorigenesis via modulation of β-catenin functions in cells. PMID:25149539

Retinoic acid receptor alpha drives cell cycle progression and is associated with increased sensitivity to retinoids in T-cell lymphoma.

PubMed

Wang, Xueju; Dasari, Surendra; Nowakowski, Grzegorz S; Lazaridis, Konstantinos N; Wieben, Eric D; Kadin, Marshall E; Feldman, Andrew L; Boddicker, Rebecca L

2017-04-18

Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with generally poor outcomes following standard therapy. Few candidate therapeutic targets have been identified to date. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to retinoids. While retinoids have been used to treat some cutaneous T-cell lymphomas (CTCLs), their mechanism of action and the role of RARA in CTCL and other mature T-cell lymphomas remain poorly understood. After identifying a PTCL with a RARAR394Q mutation, we sought to characterize the role of RARA in T-cell lymphoma cells. Overexpressing wild-type RARA or RARAR394Q significantly increased cell growth in RARAlow cell lines, while RARA knockdown induced G1 arrest and decreased expression of cyclin-dependent kinases CDK2/4/6 in RARAhigh cells. The retinoids, AM80 (tamibarotene) and all-trans retinoic acid, caused dose-dependent growth inhibition, G1 arrest, and CDK2/4/6 down-regulation. Genes down-regulated in transcriptome data were enriched for cell cycle and G1-S transition. Finally, RARA overexpression augmented chemosensitivity to retinoids. In conclusion, RARA drives cyclin-dependent kinase expression, G1-S transition, and cell growth in T-cell lymphoma. Synthetic retinoids inhibit these functions in a dose-dependent fashion and are most effective in cells with high RARA expression, indicating RARA may represent a therapeutic target in some PTCLs.

K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression.

PubMed

Hao, Zhenyue; Sheng, Yi; Duncan, Gordon S; Li, Wanda Y; Dominguez, Carmen; Sylvester, Jennifer; Su, Yu-Wen; Lin, Gloria H Y; Snow, Bryan E; Brenner, Dirk; You-Ten, Annick; Haight, Jillian; Inoue, Satoshi; Wakeham, Andrew; Elford, Alisha; Hamilton, Sara; Liang, Yi; Zúñiga-Pflücker, Juan C; He, Housheng Hansen; Ohashi, Pamela S; Mak, Tak W

2017-01-13

T-cell proliferation is regulated by ubiquitination but the underlying molecular mechanism remains obscure. Here we report that Lys-48-linked ubiquitination of the transcription factor KLF4 mediated by the E3 ligase Mule promotes T-cell entry into S phase. Mule is elevated in T cells upon TCR engagement, and Mule deficiency in T cells blocks proliferation because KLF4 accumulates and drives upregulation of its transcriptional targets E2F2 and the cyclin-dependent kinase inhibitors p21 and p27. T-cell-specific Mule knockout (TMKO) mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), show impaired generation of antigen-specific CD8 + T cells with reduced cytokine production, and fail to clear LCMV infections. Thus, Mule-mediated ubiquitination of the novel substrate KLF4 regulates T-cell proliferation, autoimmunity and antiviral immune responses in vivo.

An Alternative Splice Product of IκB Kinase (IKKγ), IKKγ-Δ, Differentially Mediates Cytokine and Human T-Cell Leukemia Virus Type 1 Tax-Induced NF-κB Activation

PubMed Central

Hai, Tao; Yeung, Man-Lung; Wood, Thomas G.; Wei, Yuanfen; Yamaoka, Shoji; Gatalica, Zoran; Jeang, Kuan-Teh; Brasier, Allan R.

2006-01-01

NF-κB is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IκB kinase (IKK) “signalsome”. The core IKK consists of two catalytic serine kinases, IKKα and IKKβ, and a noncatalytic subunit, IKKγ. IKKγ is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKKγ mRNA isoform, encoding an in-frame deletion of exon 5, termed IKKγ-Δ. Using a specific reverse transcription-PCR assay, we find that IKKγ-Δ is widely expressed in cultured human cells and normal human tissues. Because IKKγ-Δ protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax-induced NF-κB activity. Coimmunoprecipitation and confocal colocalization assays indicate IKKγ-Δ has strong homo- and heterotypic association with wild-type (WT) IKKγ and, like IKKγ WT, associates with the IKKβ kinase. Similarly, IKKγ-Δ mediates IKK kinase activity and downstream NF-κB-dependent transcription in response to tumor necrosis factor (TNF) and the NF-κB-inducing kinase-IKKα signaling pathway. Surprisingly, however, in contrast to IKKγ WT, IKKγ-Δ is not able to mediate HTLV-1 Tax-induced NF-κB-dependent transcription, even though IKKγ-Δ binds and colocalizes with Tax. These observations suggest that IKKγ-Δ is a functionally distinct alternatively spliced mRNA product differentially mediating TNF-induced, but not Tax-induced, signals converging on the IKK signalsome. Differing levels of IKKγ-Δ expression, therefore, may affect signal transduction cascades coupling to IKK. PMID:16611882

T-cell suicide gene therapy prompts thymic renewal in adults after hematopoietic stem cell transplantation.

PubMed

Vago, Luca; Oliveira, Giacomo; Bondanza, Attilio; Noviello, Maddalena; Soldati, Corrado; Ghio, Domenico; Brigida, Immacolata; Greco, Raffaella; Lupo Stanghellini, Maria Teresa; Peccatori, Jacopo; Fracchia, Sergio; Del Fiacco, Matteo; Traversari, Catia; Aiuti, Alessandro; Del Maschio, Alessandro; Bordignon, Claudio; Ciceri, Fabio; Bonini, Chiara

2012-08-30

The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially incompatible hematopoietic stem cell transplantation (HSCT). In the TK007 clinical trial, 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the HSV thymidine kinase suicide gene (TK+ cells). After a first wave of circulating TK+ cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naive lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK+ -cell engraftment. Accordingly, after the infusions, we documented an increase in circulating TCR excision circles and CD31+ recent thymic emigrants and a substantial expansion of the active thymic tissue as shown by chest tomography scans. Interestingly, a peak in the serum level of IL-7 was observed after each infusion of TK+ cells, anticipating the appearance of newly generated T cells. The results of the present study show that the infusion of genetically modified donor T cells after HSCT can drive the recovery of thymic activity in adults, leading to immune reconstitution.

Targeting the Golgi apparatus to overcome acquired resistance of non-small cell lung cancer cells to EGFR tyrosine kinase inhibitors

PubMed Central

Katayama, Ryohei; Fang, Siyang; Tsutsui, Saki; Akatsuka, Akinobu; Shan, Mingde; Choi, Hyeong-Wook; Fujita, Naoya; Yoshimatsu, Kentaro; Shiina, Isamu; Yamori, Takao; Dan, Shingo

2018-01-01

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKIs) were demonstrated to provide survival benefit in patients with non-small cell lung cancer (NSCLC) harboring activating mutations of EGFR; however, emergence of acquired resistance to EGFR-TKIs has been shown to cause poor outcome. To overcome the TKI resistance, drugs with different mode of action are required. We previously reported that M-COPA (2-methylcoprophilinamide), a Golgi disruptor, suppressed the growth of gastric cancers overexpressing receptor tyrosine kinases (RTKs) such as hepatocyte growth factor receptor (MET) via downregulating their cell surface expression. In this study, we examined the antitumor effect of M-COPA on NSCLC cells with TKI resistance. As a result, M-COPA effectively downregulated cell surface EGFR and its downstream signals, and finally exerted in vivo antitumor effect in NSCLC cells harboring secondary (T790M/del19) and tertiary (C797S/T790M/del19) mutated EGFR, which exhibit acquired resistance to first- and third generation EGFR-TKIs, respectively. M-COPA also downregulated MET expression potentially involved in the acquired resistance to EGFR-TKIs via bypassing the EGFR pathway blockade. These results provide the first evidence that targeting the Golgi apparatus might be a promising therapeutic strategy to overcome the vicious cycle of TKI resistance in EGFR-mutated NSCLC cells via downregulating cell surface RTK expression. PMID:29416720

Activated phosphoinositide 3-kinase δ syndrome presenting with gut-associated T-cell lymphoproliferative disease.

PubMed

Teranishi, Hideto; Ishimura, Masataka; Koga, Yuuki; Eguchi, Katsuhide; Sonoda, Motoshi; Kobayashi, Tetsuko; Shiraishi, Satoru; Nakashima, Kentaro; Ikegami, Kouji; Aman, Murasaki; Yamamoto, Hidetaka; Takada, Hidetoshi; Ohga, Shouichi

2017-01-01

A 13-year-old boy was admitted to our hospital because of persistent diarrhea, abdominal pain, and bloody stools. The patient had experienced repeated hospitalizations for the treatment of respiratory infections since early childhood. Colonoscopic and pathological studies led to a diagnosis of gut-associated T-cell lymphoproliferative disease (T-cell LPD). Laboratory data showed T-lymphocytopenia (492/µl), increased serum IgG levels (1,984 mg/dl), and low serum antibody titers for specific pathogens. Combined immunodeficiency accompanied by T-LPD suggested the diagnosis of activated PI3Kδ syndrome (APDS). Genetic analyses identified a heterozygous mutation of the PIK3CD gene (c.1573 G to A p.Glu525Lys). Although prednisolone and cyclosporine therapy has controlled the T-cell LPD, this patient awaits allogeneic hematopoietic cell transplantation to achieve a complete cure of his APDS.

Improving T-cell expansion and function for adoptive T-cell therapy using ex vivo treatment with PI3Kδ inhibitors and VIP antagonists

PubMed Central

Petersen, Christopher T.; Hassan, Mojibade; Morris, Anna B.; Jeffery, Jasmin; Lee, Kunhee; Jagirdar, Neera; Staton, Ashley D.; Raikar, Sunil S.; Spencer, Harold T.; Sulchek, Todd; Flowers, Christopher R.

2018-01-01

Adoptive therapy with ex vivo–expanded genetically modified antigen-specific T cells can induce remissions in patients with relapsed/refractory cancer. The clinical success of this therapy depends upon efficient transduction and expansion of T cells ex vivo and their homing, persistence and cytotoxicity following reinfusion. Lower rates of ex vivo expansion and clinical response using anti-CD19 chimeric antigen receptor (CAR) T cells have been seen in heavily pretreated lymphoma patients compared with B-cell acute lymphoblastic leukemia patients and motivate the development of novel strategies to enhance ex vivo T cell expansion and their persistence in vivo. We demonstrate that inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ) and antagonism of vasoactive intestinal peptide (VIP) signaling partially inhibits the terminal differentiation of T cells during anti-CD3/CD28 bead-mediated expansion (mean, 54.4% CD27+CD28+ T cells vs 27.4% in control cultures; P < .05). This strategy results in a mean of 83.7% more T cells cultured from lymphoma patients in the presence of PI3Kδ and VIP antagonists, increased survival of human T cells from a lymphoma patient in a murine xenograft model, enhanced cytotoxic activity of antigen-specific human CAR T cells and murine T cells against lymphoma, and increased transduction and expansion of anti-CD5 human CAR T cells. PI3Kδ and VIP antagonist-expanded T cells from lymphoma patients show reduced terminal differentiation, enhanced polyfunctional cytokine expression, and preservation of costimulatory molecule expression. Taken together, synergistic blockade of these pathways is an attractive strategy to enhance the expansion and functional capacity of ex vivo–expanded cancer-specific T cells. PMID:29386194

Effects of protein tyrosine phosphatase-PEST are reversed by Akt in T cells.

PubMed

Arimura, Yutaka; Shimizu, Kazuhiko; Koyanagi, Madoka; Yagi, Junji

2014-12-01

T cell activation is regulated by a balance between phosphorylation and dephosphorylation that is under the control of kinases and phosphatases. Here, we examined the role of a non-receptor-type protein tyrosine phosphatase, PTP-PEST, using retrovirus-mediated gene transduction into murine T cells. Based on observations of vector markers (GFP or Thy1.1), exogenous PTP-PEST-positive CD4(+) T cells appeared within 2 days after gene transduction; the percentage of PTP-PEST-positive cells tended to decrease during a resting period in the presence of IL-2 over the next 2 days. These vector markers also showed much lower expression intensities, compared with control cells, suggesting a correlation between the percent reduction and the low marker expression intensity. A catalytically inactive PTP-PEST mutant also showed the same tendency, and stepwise deletion mutants gradually lost their ability to induce the above phenomenon. On the other hand, these PTP-PEST-transduced cells did not have an apoptotic phenotype. No difference in the total cell numbers was found in the wells of a culture plate containing VEC- and PTP-PEST-transduced T cells. Moreover, serine/threonine kinase Akt, but not the anti-apoptotic molecules Bcl-2 and Bcl-XL, reversed the phenotype induced by PTP-PEST. We discuss the novel mechanism by which Akt interferes with PTP-PEST. Copyright © 2014 Elsevier Inc. All rights reserved.

A Novel Isoform of the B Cell Tyrosine Kinase BTK Protects Breast Cancer Cells from Apoptosis

PubMed Central

Eifert, Cheryl; Wang, Xianhui; Kokabee, Leila; Kourtidis, Antonis; Jain, Ritu; Gerdes, Michael J.; Conklin, Douglas S.

2016-01-01

Tyrosine kinases orchestrate key cellular signaling pathways and their dysregulation is often associated with cellular transformation. Several recent cases in which inhibitors of tyrosine kinases have been successfully used as anticancer agents have underscored the importance of this class of proteins in the development of targeted cancer therapies. We have carried out a large-scale loss-of-function analysis of the human tyrosine kinases using RNA interference to identify novel survival factors for breast cancer cells. In addition to kinases with known roles in breast and other cancers, we identified several kinases that were previously unknown to be required for breast cancer cell survival. The most surprising of these was the cytosolic, nonreceptor tyrosine kinase, Bruton’s tyrosine kinase (BTK), which has been extensively studied in B cell development. Down regulation of this protein with RNAi or inhibition with pharmacological inhibitors causes apoptosis; overexpression inhibits apoptosis induced by Doxorubicin in breast cancer cells. Our results surprisingly show that BTK is expressed in several breast cancer cell lines and tumors. The predominant form of BTK found in tumor cells is transcribed from an alternative promoter and results in a protein with an amino-terminal extension. This alternate form of BTK is expressed at significantly higher levels in tumorigenic breast cells than in normal breast cells. Since this protein is a survival factor for these cells, it represents both a potential marker and novel therapeutic target for breast cancer. PMID:23913792

Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations.

PubMed

Rius, Cristina; Attaf, Meriem; Tungatt, Katie; Bianchi, Valentina; Legut, Mateusz; Bovay, Amandine; Donia, Marco; Thor Straten, Per; Peakman, Mark; Svane, Inge Marie; Ott, Sascha; Connor, Tom; Szomolay, Barbara; Dolton, Garry; Sewell, Andrew K

2018-04-01

Peptide-MHC (pMHC) multimers, usually used as streptavidin-based tetramers, have transformed the study of Ag-specific T cells by allowing direct detection, phenotyping, and enumeration within polyclonal T cell populations. These reagents are now a standard part of the immunology toolkit and have been used in many thousands of published studies. Unfortunately, the TCR-affinity threshold required for staining with standard pMHC multimer protocols is higher than that required for efficient T cell activation. This discrepancy makes it possible for pMHC multimer staining to miss fully functional T cells, especially where low-affinity TCRs predominate, such as in MHC class II-restricted responses or those directed against self-antigens. Several recent, somewhat alarming, reports indicate that pMHC staining might fail to detect the majority of functional T cells and have prompted suggestions that T cell immunology has become biased toward the type of cells amenable to detection with multimeric pMHC. We use several viral- and tumor-specific pMHC reagents to compare populations of human T cells stained by standard pMHC protocols and optimized protocols that we have developed. Our results confirm that optimized protocols recover greater populations of T cells that include fully functional T cell clonotypes that cannot be stained by regular pMHC-staining protocols. These results highlight the importance of using optimized procedures that include the use of protein kinase inhibitor and Ab cross-linking during staining to maximize the recovery of Ag-specific T cells and serve to further highlight that many previous quantifications of T cell responses with pMHC reagents are likely to have considerably underestimated the size of the relevant populations. Copyright © 2018 The Authors.

Regulation of Asymmetric Division by Atypical Protein Kinase C Influences Early Specification of CD8+ T Lymphocyte Fates

PubMed Central

Metz, Patrick J.; Lopez, Justine; Kim, Stephanie H.; Akimoto, Kazunori; Ohno, Shigeo; Chang, John T.

2016-01-01

Naïve CD8+ T lymphocytes responding to microbial pathogens give rise to effector T cells that provide acute defense and memory T cells that provide long-lived immunity. Upon activation, CD8+ T lymphocytes can undergo asymmetric division, unequally distributing factors to the nascent daughter cells that influence their eventual fate towards the effector or memory lineages. Individual loss of either atypical protein kinase C (aPKC) isoform, PKCζ or PKCλ/ι, partially impairs asymmetric divisions and increases CD8+ T lymphocyte differentiation toward a long-lived effector fate at the expense of memory T cell formation. Here, we show that deletion of both aPKC isoforms resulted in a deficit in asymmetric divisions, increasing the proportion of daughter cells that inherit high amounts of effector fate-associated molecules, IL-2Rα, T-bet, IFNγR, and interferon regulatory factor 4 (IRF4). However, unlike CD8+ T cells deficient in only one aPKC isoform, complete loss of aPKC unexpectedly increased CD8+ T cell differentiation toward a short-lived, terminal effector fate, as evidenced by increased rates of apoptosis and decreased expression of Eomes and Bcl2 early during the immune response. Together, these results provide evidence for an important role for asymmetric division in CD8+ T lymphocyte fate specification by regulating the balance between effector and memory precursors at the initiation of the adaptive immune response. PMID:26765121

Development of highly sensitive cell-based AKT kinase ELISA for monitoring PI3K beta activity and compound efficacy.

PubMed

Yanamandra, Mahesh; Kole, Labanyamoy; Giri, Archana; Mitra, Sayan

2017-01-01

Phosphatidylinositol-3 kinase (PI3K) pathway regulates multiple cellular functions involving cell survival, growth, motility proliferation, apoptosis, and adhesion. These are deregulated in various diseases such as cancer, atherosclerosis, and inflammation. PI3Ks phosphorylate phosphatidylinositol 4,5-biphosphate (PIP2) yielding phosphatidylinositol 3, 4, 5 triphosphate (PIP3) which in turn activate AKT kinase (serine/threonine kinase), the central enzyme in regulation of metabolic functions. Due to their implications in disease pathophysiology, PI3K/AKT inhibitors became attractive targets for pharmaceutical industries. In order to assess the functional response generated by PI3K inhibitors, an appropriate cell-based screening system is essential in any screening cascade. Here we report the development of highly sensitive in-vitro cell-based kinase ELISA which quantifies the phosphorylated AKT kinase (serine 473) and total AKT kinase directly within the cells upon compound treatment. PI3Kβ overexpressing NIH3T3 cells stimulated by lysophosphatidic acid was used for PI3K/Akt pathway activation. Assay performance reliability and robustness were determined by percentage coefficient of variation (%CV) and Z factor which demonstrated an excellent agreement with assay guidelines. This 96-well plate medium throughput assay methodology was used to screen novel molecules and proved a commendable tool to study the mechanism of action property and target engagement of novel PI3K inhibitors in drug discovery.

Phase II study of alisertib, a selective Aurora A kinase inhibitor, in relapsed and refractory aggressive B- and T-cell non-Hodgkin lymphomas.

PubMed

Friedberg, Jonathan W; Mahadevan, Daruka; Cebula, Erin; Persky, Daniel; Lossos, Izidore; Agarwal, Amit B; Jung, Jungah; Burack, Richard; Zhou, Xiaofei; Leonard, E Jane; Fingert, Howard; Danaee, Hadi; Bernstein, Steven H

2014-01-01

Aurora A kinase (AAK) is overexpressed in aggressive lymphomas and can correlate with more histologically aggressive forms of disease. We therefore designed a phase II study of alisertib, a selective AAK inhibitor, in patients with relapsed and refractory aggressive non-Hodgkin lymphomas. Patients age ≥ 18 years were eligible if they had relapsed or refractory diffuse large B-cell lymphoma (DLBCL), mantle-cell lymphoma (MCL), transformed follicular lymphoma, Burkitt's lymphoma, or noncutaneous T-cell lymphoma. Alisertib was administered orally at 50 mg twice daily for 7 days in 21-day cycles. We enrolled 48 patients. Histologies included DLBCL (n = 21), MCL (n = 13), peripheral T-cell lymphoma (n = 8), transformed follicular lymphoma (n = 5), and Burkitt's (n = 1). Most common grade 3 to 4 adverse events were neutropenia (63%), leukopenia (54%), anemia (35%), thrombocytopenia (33%), stomatitis (15%), febrile neutropenia (13%), and fatigue (6%). Four deaths during the study were attributed to progressive non-Hodgkin lymphoma (n = 2), treatment-related sepsis (n = 1), and unknown cause (n = 1). The overall response rate was 27%, including responses in three of 21 patients with DLBCL, three of 13 with MCL, one of one with Burkitt's lymphoma, two of five with transformed follicular lymphoma, and four of eight with noncutaneous T-cell lymphoma. The alisertib steady-state trough concentration (n = 25) revealed the expected pharmacokinetic variability, with a trend for higher incidence of adverse event-related dose reductions at higher trough concentrations. Analysis for AAK gene amplification and total AAK protein revealed no differences between histologies or correlation with clinical response. The novel AAK inhibitor alisertib seems clinically active in both B- and T-cell aggressive lymphomas. On the basis of these results, confirmatory single-agent and combination studies have been initiated.

Phase II Study of Alisertib, a Selective Aurora A Kinase Inhibitor, in Relapsed and Refractory Aggressive B- and T-Cell Non-Hodgkin Lymphomas

PubMed Central

Friedberg, Jonathan W.; Mahadevan, Daruka; Cebula, Erin; Persky, Daniel; Lossos, Izidore; Agarwal, Amit B.; Jung, JungAh; Burack, Richard; Zhou, Xiaofei; Leonard, E. Jane; Fingert, Howard; Danaee, Hadi; Bernstein, Steven H.

2014-01-01

Purpose Aurora A kinase (AAK) is overexpressed in aggressive lymphomas and can correlate with more histologically aggressive forms of disease. We therefore designed a phase II study of alisertib, a selective AAK inhibitor, in patients with relapsed and refractory aggressive non-Hodgkin lymphomas. Patients and Methods Patients age ≥ 18 years were eligible if they had relapsed or refractory diffuse large B-cell lymphoma (DLBCL), mantle-cell lymphoma (MCL), transformed follicular lymphoma, Burkitt's lymphoma, or noncutaneous T-cell lymphoma. Alisertib was administered orally at 50 mg twice daily for 7 days in 21-day cycles. Results We enrolled 48 patients. Histologies included DLBCL (n = 21), MCL (n = 13), peripheral T-cell lymphoma (n = 8), transformed follicular lymphoma (n = 5), and Burkitt's (n = 1). Most common grade 3 to 4 adverse events were neutropenia (63%), leukopenia (54%), anemia (35%), thrombocytopenia (33%), stomatitis (15%), febrile neutropenia (13%), and fatigue (6%). Four deaths during the study were attributed to progressive non-Hodgkin lymphoma (n = 2), treatment-related sepsis (n = 1), and unknown cause (n = 1). The overall response rate was 27%, including responses in three of 21 patients with DLBCL, three of 13 with MCL, one of one with Burkitt's lymphoma, two of five with transformed follicular lymphoma, and four of eight with noncutaneous T-cell lymphoma. The alisertib steady-state trough concentration (n = 25) revealed the expected pharmacokinetic variability, with a trend for higher incidence of adverse event–related dose reductions at higher trough concentrations. Analysis for AAK gene amplification and total AAK protein revealed no differences between histologies or correlation with clinical response. Conclusion The novel AAK inhibitor alisertib seems clinically active in both B- and T-cell aggressive lymphomas. On the basis of these results, confirmatory single-agent and combination studies have been initiated. PMID:24043741

Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal

PubMed Central

Kinehara, Masaki; Kawamura, Suguru; Tateyama, Daiki; Suga, Mika; Matsumura, Hiroko; Mimura, Sumiyo; Hirayama, Noriko; Hirata, Mitsuhi; Uchio-Yamada, Kozue; Kohara, Arihiro; Yanagihara, Kana; Furue, Miho K.

2013-01-01

Background The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. Methodology/Principal Findings In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells. Conclusions/Significance Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long

EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma.

PubMed

Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

2016-01-01

Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase

EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma

PubMed Central

Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

2016-01-01

Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase

Increased T-helper 17 cell differentiation mediated by exosome-mediated microRNA-451 redistribution in gastric cancer infiltrated T cells.

PubMed

Liu, Feng; Bu, Zhouyan; Zhao, Feng; Xiao, Daping

2018-01-01

MicroRNA (miR)-451 is a cell metabolism-related miRNA that can mediate cell energy-consuming models by several targets. As miR-451 can promote mechanistic target of rapamycin (mTOR) activity, and increased mTOR activity is related to increased differentiation of T-helper 17 (Th17) cells, we sought to investigate whether miR-451 can redistribute from cancer cells to infiltrated T cells and enhance the distribution of Th17 cells through mTOR. Real-time PCR was used for detecting expression of miR-451 in gastric cancer, tumor infiltrated T cells and exosomes, and distribution of Th17 was evaluated by both flow cytometry and immunohistochemistry (IHC). Immunofluorescence staining was used in monitoring the exosome-enveloped miR-451 from cancer cells to T cells with different treatments, and signaling pathway change was analyzed by western blot. miR-451 decreased significantly in gastric cancer (GC) tissues but increased in infiltrated T cells and exosomes; tumor miR-451 was negatively related to infiltrated T cells and exosome miR-451. Exosome miR-451 can not only serve as an indicator for poor prognosis of post-operation GC patients but is also related to increased Th17 distribution in gastric cancer. miR-451 can redistribute from cancer cells to T cells with low glucose treatment. Decreased 5' AMP-activated protein kinase (AMPK) and increased mTOR activity was investigated in miR-451 redistributed T cells and the Th17 polarized differentiation of these T cells were also increased. Exosome miR-451 derived from tumor tissues can serve as an indicator for poor prognosis and redistribution of miR-451 from cancer cells to infiltrated T cells in low glucose treatment can enhance Th17 differentiation by enhancing mTOR activity. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

The Visible Human Data Sets (VHD) and Insight Toolkit (ITk): Experiments in Open Source Software

PubMed Central

Ackerman, Michael J.; Yoo, Terry S.

2003-01-01

From its inception in 1989, the Visible Human Project was designed as an experiment in open source software. In 1994 and 1995 the male and female Visible Human data sets were released by the National Library of Medicine (NLM) as open source data sets. In 2002 the NLM released the first version of the Insight Toolkit (ITk) as open source software. PMID:14728278

Bacterial superantigens bypass Lck-dependent T cell receptor signaling by activating a Galpha11-dependent, PLC-beta-mediated pathway.

PubMed

Bueno, Clara; Lemke, Caitlin D; Criado, Gabriel; Baroja, Miren L; Ferguson, Stephen S G; Rahman, A K M Nur-Ur; Tsoukas, Constantine D; McCormick, John K; Madrenas, Joaquin

2006-07-01

The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.

Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes

PubMed Central

2012-01-01

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. PMID:22747577

MEK inhibition prevents tumour-shed transforming growth factor-β-induced T-regulatory cell augmentation in tumour milieu.

PubMed

Hossain, Dewan M S; Panda, Abir K; Chakrabarty, Sreeparna; Bhattacharjee, Pushpak; Kajal, Kirti; Mohanty, Suchismita; Sarkar, Irene; Sarkar, Diptendra K; Kar, Santosh K; Sa, Gaurisankar

2015-04-01

Tumour progression is associated with immune-suppressive conditions that facilitate the escape of tumour cells from the regimen of immune cells, subsequently paralysing the host defence mechanisms. Induction of CD4(+)  CD25(+)  FoxP3(+) T regulatory (Treg) cells has been implicated in the tumour immune escape mechanism, although the novel anti-cancer treatment strategies targeting Treg cells remain unknown. The focus of this study is to define the interaction between tumour and immune system, i.e. how immune tolerance starts and gradually leads to the induction of adaptive Treg cells in the tumour microenvironment. Our study identified hyperactivated mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) -signalling as a potential target for reversing Treg cell augmentation in breast cancer patients. In more mechanistic detail, pharmacological inhibitors of MEK/ERK signalling inhibited transforming growth factor-β (TGF-β) production in tumour cells that essentially blocked TGF-β-SMAD3/SMAD4-mediated induction of CD25/interleukin-2 receptor α on CD4(+) T-cell surface. As a result high-affinity binding of interleukin-2 on those cells was prohibited, causing lack of Janus kinase 1 (JAK1)/JAK3-mediated signal transducer and activator of transcription 3 (STAT3)/STAT5 activation required for FoxP3 expression. Finally, for a more radical approach towards a safe MEK inhibitor, we validate the potential of multi-kinase inhibitor curcumin, especially the nano-curcumin made out of pure curcumin with greater bioavailability; in repealing tumour-shed TGF-β-induced Treg cell augmentation. © 2014 Bose Institute.

MEK inhibition prevents tumour-shed transforming growth factor-β-induced T-regulatory cell augmentation in tumour milieu

PubMed Central

Hossain, Dewan M S; Panda, Abir K; Chakrabarty, Sreeparna; Bhattacharjee, Pushpak; Kajal, Kirti; Mohanty, Suchismita; Sarkar, Irene; Sarkar, Diptendra K; Kar, Santosh K; Sa, Gaurisankar

2015-01-01

Tumour progression is associated with immune-suppressive conditions that facilitate the escape of tumour cells from the regimen of immune cells, subsequently paralysing the host defence mechanisms. Induction of CD4+ CD25+ FoxP3+ T regulatory (Treg) cells has been implicated in the tumour immune escape mechanism, although the novel anti-cancer treatment strategies targeting Treg cells remain unknown. The focus of this study is to define the interaction between tumour and immune system, i.e. how immune tolerance starts and gradually leads to the induction of adaptive Treg cells in the tumour microenvironment. Our study identified hyperactivated mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) -signalling as a potential target for reversing Treg cell augmentation in breast cancer patients. In more mechanistic detail, pharmacological inhibitors of MEK/ERK signalling inhibited transforming growth factor-β (TGF-β) production in tumour cells that essentially blocked TGF-β-SMAD3/SMAD4-mediated induction of CD25/interleukin-2 receptor α on CD4+ T-cell surface. As a result high-affinity binding of interleukin-2 on those cells was prohibited, causing lack of Janus kinase 1 (JAK1)/JAK3-mediated signal transducer and activator of transcription 3 (STAT3)/STAT5 activation required for FoxP3 expression. Finally, for a more radical approach towards a safe MEK inhibitor, we validate the potential of multi-kinase inhibitor curcumin, especially the nano-curcumin made out of pure curcumin with greater bioavailability; in repealing tumour-shed TGF-β-induced Treg cell augmentation. PMID:25284464

Stretch-Enhancers Delineate Disease-Associated Regulatory Nodes in T Cells

PubMed Central

Vahedi, Golnaz; Kanno, Yuka; Furumoto, Yasuko; Jiang, Kan; Parker, Stephen C.; Erdos, Michael; Davis, Sean R.; Roychoudhuri, Rahul; Restifo, Nicholas P.; Gadina, Massimo; Tang, Zhonghui; Ruan, Yijun; Collins, Francis S.; Sartorelli, Vittorio; O’Shea, John J.

2014-01-01

Enhancers regulate spatiotemporal gene expression and impart cell-specific transcriptional outputs that drive cell identity1. Stretch- or super-enhancers (SEs) are a subset of enhancers especially important for genes associated with cell identity and genetic risk of disease2,3,4,5,6. CD4+ T cells are critical for host defense and autoimmunity. Herein, we analyzed maps of T cell SEs as a non-biased means of identifying key regulatory nodes involved in cell specification. We found that cytokines and cytokine receptors were the dominant class of genes exhibiting SE architecture in T cells. This notwithstanding, the locus encoding Bach2, a key negative regulator of effector differentiation, emerged as the most prominent T cell SE, revealing a network wherein SE-associated genes critical for T cell biology are repressed by BACH2. Disease-associated SNPs for immune-mediated disorders, including rheumatoid arthritis (RA), were highly enriched for T cell-SEs versus typical enhancers (TEs) or SEs in other cell lineages7. Intriguingly, treatment of T cells with the Janus kinase (JAK) inhibitor, tofacitinib, disproportionately altered the expression of RA risk genes with SE structures. Together, these results indicate that genes with SE architecture in T cells encompass a variety of cytokines and cytokine receptors but are controlled by a “guardian” transcription factor, itself endowed with an SE. Thus, enumeration of SEs allows unbiased determination of key regulatory nodes in T cells, which are preferentially modulated by pharmacological intervention. PMID:25686607

Functional heterogeneity of human effector CD8+ T cells.

PubMed

Takata, Hiroshi; Naruto, Takuya; Takiguchi, Masafumi

2012-02-09

Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

Focal adhesion kinase

PubMed Central

Stone, Rebecca L; Baggerly, Keith A; Armaiz-Pena, Guillermo N; Kang, Yu; Sanguino, Angela M; Thanapprapasr, Duangmani; Dalton, Heather J; Bottsford-Miller, Justin; Zand, Behrouz; Akbani, Rehan; Diao, Lixia; Nick, Alpa M; DeGeest, Koen; Lopez-Berestein, Gabriel; Coleman, Robert L; Lutgendorf, Susan; Sood, Anil K

2014-01-01

This investigation describes the clinical significance of phosphorylated focal adhesion kinase (FAK) at the major activating tyrosine site (Y397) in epithelial ovarian cancer (EOC) cells and tumor-associated endothelial cells. FAK gene amplification as a mechanism for FAK overexpression and the effects of FAK tyrosine kinase inhibitor VS-6062 on tumor growth, metastasis, and angiogenesis were examined. FAK and phospho-FAKY397 were quantified in tumor (FAK-T; pFAK-T) and tumor-associated endothelial (FAK-endo; pFAK-endo) cell compartments of EOCs using immunostaining and qRT-PCR. Associations between expression levels and clinical variables were evaluated. Data from The Cancer Genome Atlas were used to correlate FAK gene copy number and expression levels in EOC specimens. The in vitro and in vivo effects of VS-6062 were assayed in preclinical models. FAK-T and pFAK-T overexpression was significantly associated with advanced stage disease and increased microvessel density (MVD). High MVD was observed in tumors with elevated endothelial cell FAK (59%) and pFAK (44%). Survival was adversely affected by FAK-T overexpression (3.03 vs 2.06 y, P = 0.004), pFAK-T (2.83 vs 1.78 y, P < 0.001), and pFAK-endo (2.33 vs 2.17 y, P = 0.005). FAK gene copy number was increased in 34% of tumors and correlated with expression levels (P < 0.001). VS-6062 significantly blocked EOC and endothelial cell migration as well as endothelial cell tube formation in vitro. VS-6062 reduced mean tumor weight by 56% (P = 0.005), tumor MVD by 40% (P = 0.0001), and extraovarian metastasis (P < 0.01) in orthotopic EOC mouse models. FAK may be a unique therapeutic target in EOC given the dual anti-angiogenic and anti-metastatic potential of FAK inhibitors. PMID:24755674

Activated type I TGFbeta receptor (Alk5) kinase confers enhancedsurvival to mammary epithelial cells and accelerates mammary tumorprogression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muraoka-Cook, Rebecca S.; Shin, Incheol; Yi, Jae Youn

2005-01-02

The transforming growth factor-betas (TGF{beta}s) are members of a large superfamily of pleiotropic cytokines that also includes the activins and the bone morphogenetic proteins (BMPs). Members of the TGF{beta} family regulate complex physiological processes such cell proliferation, differentiation, adhesion, cell-cell and cell-matrix interactions, motility, and cell death, among others (Massague, 1998). Dysregulation of TGF{beta} signaling contributes to several pathological processes including cancer, fibrosis, and auto-immune disorders (Massague et al., 2000). The TGF{beta}s elicit their biological effects by binding to type II and type I transmembrane receptor serine-threonine kinases (T{beta}RII and T{beta}RI) which, in turn, phosphorylated Smad 2 and Smad 3.more » Phosphorylated Smad 2/3 associate with Smad 4 and, as a heteromeric complex, translocate to the nucleus where they regulate gene transcription. The inhibitory Smad7 down regulates TGF{beta} signaling by binding to activated T{beta}RI and interfering with its ability to phosphorylate Smad 2/3 (Derynck and Zhang, 2003; Shi and Massague, 2003). Signaling is also regulated by Smad proteolysis. TGF{beta} receptor-mediated activation results in multi-ubiquitination of Smad 2 in the nucleus and subsequent degradation of Smad 2 by the proteasome (Lo and Massague, 1999). Activation of TGF{beta} receptors also induces mobilization of a Smad 7-Smurf complex from the nucleus to the cytoplasm; this complex recognizes the activated receptors and mediates their ubiquitination and internalization via caveolin-rich vesicles, leading to termination of TGF{beta} signaling (Di Guglielmo et al., 2003). Other signal transducers/pathways have been implicated in TGF{beta} actions. These include the extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (Jnk), p38 mitogen-activated protein kinase (MAPK), protein phosphatase PP2A, phosphatidylinositol-3 kinase (PI3K), and the family of Rho GTPases [reviewed

The Legionella pneumophila orphan sensor kinase LqsT regulates competence and pathogen-host interactions as a component of the LAI-1 circuit.

PubMed

Kessler, Aline; Schell, Ursula; Sahr, Tobias; Tiaden, André; Harrison, Christopher; Buchrieser, Carmen; Hilbi, Hubert

2013-02-01

Legionella pneumophila is an amoeba-resistant opportunistic pathogen that performs cell-cell communication through the signalling molecule 3-hydroxypentadecane-4-one (LAI-1, Legionella autoinducer-1). The lqs (Legionella quorum sensing) gene cluster encodes the LAI-1 autoinducer synthase LqsA, the cognate sensor kinase LqsS and the response regulator LqsR. Here we show that the Lqs system includes an 'orphan' homologue of LqsS termed LqsT. Compared with wild-type L. pneumophila, strains lacking lqsT or both lqsS and lqsT show increased salt resistance, greatly enhanced natural competence for DNA acquisition and impaired uptake by phagocytes. Sensitive novel single round growth assays and competition experiments using Acanthamoeba castellanii revealed that ΔlqsT and ΔlqsS-ΔlqsT, as well as ΔlqsA and other lqs mutant strains are impaired for intracellular growth and cannot compete against wild-type bacteria upon co-infection. In contrast to the ΔlqsS strain, ΔlqsT does not produce extracellular filaments. The phenotypes of the ΔlqsS-ΔlqsT strain are partially complemented by either lqsT or lqsS, but are not reversed by overexpression of lqsA, suggesting that LqsT and LqsS are the sole LAI-1-responsive sensor kinases in L. pneumophila. In agreement with the different phenotypes of the ΔlqsT and ΔlqsS strains, lqsT and lqsS are differentially expressed in the post-exponential growth phase, and transcriptome studies indicated that 90% of the genes, which are downregulated in absence of lqsT, are upregulated in absence of lqsS. Reciprocally regulated genes encode components of a 133 kb genomic 'fitness island' or translocated effector proteins implicated in virulence. Together, these results reveal a unique organization of the L. pneumophila Lqs system comprising two partially antagonistic LAI-1-responsive sensor kinases, LqsT and LqsS, which regulate distinct pools of genes implicated in pathogen-host cell interactions, competence, expression of a

Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nobe, Koji, E-mail: kojinobe@pharm.showa-u.ac.jp; Nobe, Hiromi; Department of Physical Therapy, Bunkyo-Gakuin University

Research highlights: {yields} Mechanisms of fibroblast cell contraction in collagen matrix. {yields} Assessed an isometric force development using 3D-reconstituted-fibroblast fiber. {yields} Constitutively active Rho A induced the over-contraction of fibroblast cells. {yields} Rho A and Rho kinase pathway has a central role in fibroblast cell contraction. -- Abstract: Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibersmore » and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor ( (Y27632)) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.« less

The human tyrosine kinase Kit and its gatekeeper mutant T670I, show different kinetic properties: Implications for drug design.

PubMed

Kissova, Miroslava; Maga, Giovanni; Crespan, Emmanuele

2016-10-01

The tyrosine kinase Kit, a receptor for Stem Cell Factor, is involved, among others, in processes associated to cell survival, proliferation and migration. Upon physiological conditions, the activity of Kit is tightly regulated. However, primary mutations that lead to its constitutive activation are the causal oncogenic driver of gastrointestinal stromal tumours (GISTs). GISTs are known to be refractory to conventional therapies but the introduction of Imatinib, a selective inhibitor of tyrosine kinases Abl and Kit, significantly ameliorated the treatment options of GISTs patients. However, the acquisition of secondary mutations renders Kit resistant towards all available drugs. Mutation involving gatekeeper residues (such as V654a and T670I) influence both the structure and the catalytic activity of the enzyme. Therefore, detailed knowledge of the enzymatic properties of the mutant forms, in comparison with the wild type enzyme, is an important pre-requisite for the rational development of specific inhibitors. In this paper we report a thorough kinetic analysis of the reaction catalyzed by the Kit kinase and its gatekeeper mutated form T670I. Our results revealed the different mechanisms of action of these two enzymes and may open a new avenue for the future design of specific Kit inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aurora A kinase RNAi and small molecule inhibition of Aurora kinases with VE-465 induce apoptotic death in multiple myeloma cells.

PubMed

Evans, Robert; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Keats, Jonathan; Maxwell, Christopher; Perry, Troy; Chau, Heidi; Belch, Andrew; Pilarski, Linda; Reiman, Tony

2008-03-01

The expression of RHAMM and other centrosome-associated genes are known to correlate with the extent of centrosome amplification in multiple myeloma, and with poor prognosis. RHAMM has a significant interaction with TPX2, a protein which regulates the localization and action of Aurora A kinase (AURKA) at the spindle poles. AURKA is known to be a central determinant of centrosome and spindle function and is a target for cancer therapy. Given these observations, we investigated the role of Aurora kinases as therapeutic targets in myeloma. Here we report that AURKA is expressed ubiquitously in myeloma, to varying degrees, in both cell lines and patients' bone marrow plasma cells. siRNA targeting AURKA induces apoptotic cell death in myeloma cell lines. The Aurora kinase inhibitor VE-465 also induces apoptosis and death in myeloma cell lines and primary myeloma plasma cells. The combination of VE-465 and dexamethasone improves cell killing compared with the use of either agent alone, even in cells resistant to the single agents. The phenotype of myeloma cells treated with VE-465 is consistent with published reports on the effects of Aurora kinase inhibition. Aurora kinase inhibitors should be pursued as potential treatments for myeloma.

Inactivation of Mirk/Dyrk1b Kinase Targets Quiescent Pancreatic Cancer Cells *

PubMed Central

Ewton, Daina Z.; Hu, Jing; Vilenchik, Maria; Deng, Xiaobing; Luk, Kin-chun; Polonskaia, Ann; Hoffman, Ann F.; Zipf, Karen; Boylan, John F.; Friedman, Eileen A.

2011-01-01

A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they re-enter the cell cycle. Earlier studies demonstrated that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G0 tumor cells, that Mirk uses several mechanisms to block cell cycling, and that Mirk increases expression of antioxidant genes which lower ROS levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G0 state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1 and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In prior studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase. PMID:21878655

Effects of phorbol ester on mitogen-activated protein kinase kinase activity in wild-type and phorbol ester-resistant EL4 thymoma cells.

PubMed

Gause, K C; Homma, M K; Licciardi, K A; Seger, R; Ahn, N G; Peterson, M J; Krebs, E G; Meier, K E

1993-08-05

Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.

Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nifuji, Akira, E-mail: nifuji-a@tsurumi-u.ac.jp; Department of Pharmacology, Tsurumi University School of Dental Medicine, Yokohama; Ideno, Hisashi

2010-04-15

Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLKmore » in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.« less

Tropomyosin Receptor Kinase A Expression on Merkel Cell Carcinoma Cells.

PubMed

Wehkamp, Ulrike; Stern, Sophie; Krüger, Sandra; Hauschild, Axel; Röcken, Christoph; Egberts, Friederike

2017-11-01

Merkel cell carcinoma (MCC) is a malignant neuroendocrine skin tumor frequently associated with the Merkel cell polyomavirus. Immune checkpoint therapy showed remarkable results, although not all patients are responsive to this therapy. Anti-tropomyosin receptor kinase A (TrkA)-targeted treatment has shown promising results in several tumor entities. To determine TrkA expression in MCC as a rationale for potential targeted therapy. This case series study investigated the MCC specimens of 55 patients treated at the Department of Dermatology, University Hospital of Schleswig-Holstein, Kiel, Germany, from January 1, 2005, through December 31, 2015. Thirty-nine of the 55 samples were suitable for further histopathologic examination. Expression of TrkA was explored by immunohistochemical analysis. Diagnosis of MCC was confirmed by staining positive for cytokeratin 20 (CK20) and synaptophysin. Expression of TrkA on the tumor cells. Specimens of 39 patients (21 women and 18 men; mean [SD] age, 75.0 [7.8] years) underwent immunohistochemical investigation. Thirty-eight of 38 specimens expressed CK20 and synaptophysin on the MCC tumor cells (100% expression). Merkel cell polyomavirus was detected in 32 of 38 specimens (84%). Tropomyosin receptor kinase A was found in all 36 evaluable specimens on the tumor cells; 34 (94%) showed a weak and 2 (6%) showed a strong cytoplasmic expression. In addition, strongly positive perinuclear dots were observed in 30 of 36 specimens (83%). Tropomyosin receptor kinase A was expressed on MCC tumor cells in 100% of evaluable specimens. This result may lead to the exploration of new targeted treatment options in MCC, especially for patients who do not respond to anti-programmed cell death protein 1 treatment.

Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

PubMed Central

Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

2016-01-01

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793

The Class III Kinase Vps34 Promotes T Lymphocyte Survival through Regulating IL-7Rα Surface Expression

PubMed Central

McLeod, Ian X.; Zhou, Xiang; Li, Qi-Jing; Wang, Fan; He, You-Wen

2011-01-01

IL-7Rα mediated signals are essential for naive T lymphocyte survival. Recent studies show that IL-7Rα is internalized and either recycled to cell surface or degraded. However, how the intracellular process of IL-7Rα trafficking is regulated is unclear. Here we show that Vps34, the class III phosphatidylinositol 3-kinase, plays a critical role in proper IL-7Rα intracellular trafficking. Mice lacking Vps34 in T lymphocytes had a severely reduced T lymphocyte compartment. Vps34-deficient T lymphocytes exhibit increased death and reduced IL-7Rα surface expression, though three major forms of autophagy remain intact. Intracellular IL-7Rα in normal T lymphocytes at steady-state is trafficked through either early endosome/multivesicular bodies (MVB) to the late endosome-Golgi for surface expression or to the lysosome for degradation. However, Vps34-deficient T cells have mislocalized intracellular Eea1, HRS, and Vps36 protein levels, the combined consequence of which is the inability to mobilize internalized IL-7Rα into the retromer pathway for surface display. Our studies reveal that Vps34, though dispensible for autophagy induction, is a critical regulator of naïve T cell homeostasis, modulating IL-7Rα trafficking, signaling, and recycling. PMID:22021616

T-lymphokine-activated killer cell-originated protein kinase (TOPK) as a prognostic factor and a potential therapeutic target in glioma

PubMed Central

Duan, Qiuhong; Yuan, Ping; Xue, Peipei; Lu, Hui; Yan, Meng; Guo, Dongsheng; Xu, Sanpeng; Zhang, Xiaohui; Lin, Xuan; Wang, Yong; Dogan, Soner; Zhang, Jianmin; Zhu, Feng; Ke, Changshu; Liu, Lin

2018-01-01

TOPK is overexpressed in various types of cancer and associated with poor outcomes in different types of cancer. In this study, we first found that the expression of T-lymphokine-activated killer cell-originated protein kinase (TOPK) was significantly higher in Grade III or Grade IV than that in Grade II in glioma (P = 0.007 and P < 0.001, respectively). Expression of TOPK was positively correlated with Ki67 (P < 0.001). Knockdown of TOPK significantly inhibited cell growth, colony formation and increased sensitivities to temozolomide (TMZ) in U-87 MG or U-251 cells, while TOPK overexpression promoted cell growth and colony formation in Hs 683 or A-172 cells. Glioma patients expressing high levels of TOPK have poor survival compared with those expressing low levels of TOPK in high-grade or low-grade gliomas (hazard ratio = 0.2995; 95% CI, 0.1262 to 0.7108; P = 0.0063 and hazard ratio = 0.1509; 95% CI, 0.05928 to 0.3842; P < 0.0001, respectively). The level of TOPK was low in TMZ-sensitive patients compared with TMZ-resistant patients (P = 0.0056). In TMZ-resistant population, patients expressing high TOPK have two months’ shorter survival time than those expressing low TOPK. Our findings demonstrated that TOPK might represent as a promising prognostic and predictive factor and potential therapeutic target for glioma. PMID:29487691

Regulatory T cells control HIV replication in activated T cells through a cAMP-dependent mechanism

PubMed Central

Moreno-Fernandez, Maria E.; Rueda, Cesar Mauricio; Rusie, Laura K.

2011-01-01

We hypothesized that regulatory T cells (Tregs) could play a beneficial role during HIV infection by controlling HIV replication in conventional T cells (Tcons). Purified Tregs and Tcons from healthy donors were activated separately. Tcons were infected with the X4 or R5 HIV strains and cultured with or without autologous Tregs. Coculture of Tcons and Tregs resulted in a dose-dependent inhibition of Tcon infection, which was significant when a 1:1 Treg:Tcon ratio was used. Treg suppression of HIV infection was largely mediated by contact-dependent mechanisms. Blockage of cytotoxic T-lymphocyte–associated antigen-4 did not significantly reduce Treg function. In contrast, Tregs acted through cAMP-dependent mechanisms, because the decrease of cAMP levels in Tregs, the blockade of gap junction formation between Tregs and Tcons, the blockage of CD39 activity, and the blockage of protein kinase A in Tcons all abolished Treg-mediated suppression of HIV replication. Our data suggest a complex role for Tregs during HIV infection. Although Tregs inhibit specific immune responses, their inhibition of HIV replication in Tcons may play a beneficial role, particularly during early HIV infection, when the effector immune cells are not yet activated. Such a protective role of Tregs could have a profound impact on infection outcome. PMID:21436067

Role of nongenomic activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 pathways in 1,25D3-mediated apoptosis in squamous cell carcinoma cells.

PubMed

Ma, Yingyu; Yu, Wei-Dong; Kong, Rui-Xian; Trump, Donald L; Johnson, Candace S

2006-08-15

Vitamin D is a steroid hormone that regulates calcium homeostasis and bone metabolism. The active form of vitamin D [1 alpha,25-dihydroxyvitamin D(3) (1,25D3)] acts through both genomic and nongenomic pathways. 1,25D3 has antitumor effects in a variety of cancers, including colorectal, prostate, breast, ovarian, and skin cancers. 1,25D3 exerts growth-inhibitory effects in cancer cells through the induction of apoptosis, cell cycle arrest, and differentiation. The mechanisms regulating 1,25D3-induced apoptosis remain unclear. We investigated the role of nongenomic signaling in 1,25D3-mediated apoptosis in squamous cell carcinoma (SCC) cells. 1,25D3 induced rapid and sustained activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathways in SCC cells. These effects were nongenomic: they occurred rapidly and were not inhibited by cycloheximide or actinomycin D. To examine whether the nongenomic activation of Akt and ERK1/2 plays a role in 1,25D3-mediated apoptosis, the expression of Akt or ERK1/2 was reduced by small interfering RNA (siRNA). siRNA-Akt significantly enhanced 1,25D3-induced apoptosis as indicated by increased levels of Annexin V-positive cells and increased sub-G(1) population and DNA fragmentation. In contrast, siRNA-ERK1/2 had no effects on 1,25D3-induced apoptosis. In addition, siRNA-Akt transfection followed by 1,25D3 treatment induced apoptosis much sooner than 1,25D3 alone. siRNA-Akt and 1,25D3 induced caspase-10 activation, suppressed the expression of c-IAP1 and XIAP, and promoted 1,25D3-induced caspase-3 activation. These results support a link between 1,25D3-induced nongenomic signaling and apoptosis. 1,25D3 induces the activation of phosphatidylinositol 3-kinase/Akt, which suppresses 1,25D3-mediated apoptosis and prolongs the survival of SCC cells.

Iron depletion results in Src kinase inhibition with associated cell cycle arrest in neuroblastoma cells

PubMed Central

Siriwardana, Gamini; Seligman, Paul A

2015-01-01

Iron is required for cellular proliferation. Recently, using systematic time studies of neuroblastoma cell growth, we better defined the G1 arrest caused by iron chelation to a point in mid-G1, where cyclin E protein is present, but the cyclin E/CDK2 complex kinase activity is inhibited. In this study, we again used the neuroblastoma SKNSH cells lines to pinpoint the mechanism responsible for this G1 block. Initial studies showed in the presence of DFO, these cells have high levels of p27 and after reversal of iron chelation p27 is degraded allowing for CDK2 kinase activity. The initial activation of CDK2 kinase allows cells to exit G1 and enter S phase. Furthermore, we found that inhibition of p27 degradation by DFO is directly associated with inhibition of Src kinase activity measured by lack of phosphorylation of Src at the 416 residue. Activation of Src kinase occurs very early after reversal from the DFO G1 block and is temporally associated with initiation of cellular proliferation associated with entry into S phase. For the first time therefore we show that iron chelation inhibits Src kinase activity and this activity is a requirement for cellular proliferation. PMID:25825542

Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagashima, Kumiko; Shumway, Stuart D.; Sathyanarayanan, Sriram

2013-11-20

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitivemore » kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.« less

Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation

PubMed Central

O′Flaherty, Linda; Pardo, Olivier E.; Dzien, Piotr; Phillips, Lois; Morgan, Carys; Pawade, Joya; May, Margaret T.; Sohail, Muhammad; Hetzel, Martin R.; Seckl, Michael J.; Tavaré, Jeremy M.

2014-01-01

Background Glycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC. Methods The expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture. Results Expression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes. Conclusion NSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC. PMID:25486534

PKM2-dependent metabolic reprogramming in CD4+ T cells is crucial for hyperhomocysteinemia-accelerated atherosclerosis.

PubMed

Lü, Silin; Deng, Jiacheng; Liu, Huiying; Liu, Bo; Yang, Juan; Miao, Yutong; Li, Jing; Wang, Nan; Jiang, Changtao; Xu, Qingbo; Wang, Xian; Feng, Juan

2018-06-01

Inflammation mediated by activated T cells plays an important role in the initiation and progression of hyperhomocysteinemia (HHcy)-accelerated atherosclerosis in ApoE -/- mice. Homocysteine (Hcy) activates T cells to secrete proinflammatory cytokines, especially interferon (IFN)-γ; however, the precise mechanisms remain unclear. Metabolic reprogramming is critical for T cell inflammatory activation and effector functions. Our previous study demonstrated that Hcy regulates T cell mitochondrial reprogramming by enhancing endoplasmic reticulum (ER)-mitochondria coupling. In this study, we further explored the important role of glycolysis-mediated metabolic reprogramming in Hcy-activated CD4 + T cells. Mechanistically, Hcy-activated CD4 + T cell increased the protein expression and activity of pyruvate kinase muscle isozyme 2 (PKM2), the final rate-limiting enzyme in glycolysis, via the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin signaling pathway. Knockdown of PKM2 by small interfering RNA reduced Hcy-induced CD4 + T cell IFN-γ secretion. Furthermore, we generated T cell-specific PKM2 knockout mice by crossing LckCre transgenic mice with PKM2 fl/fl mice and observed that Hcy-induced glycolysis and oxidative phosphorylation were both diminished in PKM2-deficient CD4 + T cells with reduced glucose and lipid metabolites, and subsequently reduced IFN-γ secretion. T cell-depleted apolipoprotein E-deficient (ApoE -/- ) mice adoptively transferred with PKM2-deficient CD4 + T cells, compared to mice transferred with control cells, showed significantly decreased HHcy-accelerated early atherosclerotic lesion formation. In conclusion, this work indicates that the PKM2-dependent glycolytic-lipogenic axis, a novel mechanism of metabolic regulation, is crucial for HHcy-induced CD4 + T cell activation to accelerate early atherosclerosis in ApoE -/- mice. Metabolic reprogramming is crucial for Hcy-induced CD4 + T cell inflammatory activation. Hcy activates

Distinct and Overlapping Functions of TEC Kinase and BTK in B Cell Receptor Signaling.

PubMed

de Bruijn, Marjolein J W; Rip, Jasper; van der Ploeg, Esmee K; van Greuningen, Lars W; Ta, Van T B; Kil, Laurens P; Langerak, Anton W; Rimmelzwaan, Guus F; Ellmeier, Wilfried; Hendriks, Rudi W; Corneth, Odilia B J

2017-04-15

The Tec tyrosine kinase is expressed in many cell types, including hematopoietic cells, and is a member of the Tec kinase family that also includes Btk. Although the role of Btk in B cells has been extensively studied, the role of Tec kinase in B cells remains largely unclear. It was previously shown that Tec kinase has the ability to partly compensate for loss of Btk activity in B cell differentiation, although the underlying mechanism is unknown. In this study, we confirm that Tec kinase is not essential for normal B cell development when Btk is present, but we also found that Tec-deficient mature B cells showed increased activation, proliferation, and survival upon BCR stimulation, even in the presence of Btk. Whereas Tec deficiency did not affect phosphorylation of phospholipase Cγ or Ca 2+ influx, it was associated with significantly increased activation of the intracellular Akt/S6 kinase signaling pathway upon BCR and CD40 stimulation. The increased S6 kinase phosphorylation in Tec-deficient B cells was dependent on Btk kinase activity, as ibrutinib treatment restored pS6 to wild-type levels, although Btk protein and phosphorylation levels were comparable to controls. In Tec-deficient mice in vivo, B cell responses to model Ags and humoral immunity upon influenza infection were enhanced. Moreover, aged mice lacking Tec kinase developed a mild autoimmune phenotype. Taken together, these data indicate that in mature B cells, Tec and Btk may compete for activation of the Akt signaling pathway, whereby the activating capacity of Btk is limited by the presence of Tec kinase. Copyright © 2017 by The American Association of Immunologists, Inc.

PTPROt maintains T cell immunity in the microenvironment of hepatocellular carcinoma.

PubMed

Hou, Jiajie; Deng, Lei; Zhuo, Han; Lin, Zhe; Chen, Yun; Jiang, Runqiu; Chen, Dianyu; Zhang, Xudong; Huang, Xingxu; Sun, Beicheng

2015-08-01

Intratumoral T cells play a central role in anti-tumor immunity, and the balance between T effector cells (Teff) and regulatory T cells (Treg) affects the prognosis of cancer patients. However, educated by tumor microenvironment, T cells frequently fail in their responsibility. In this study, we aimed to investigate the role of truncated isoform of protein tyrosine phosphatase receptor-type O (PTPROt) in T cell-mediated anti-tumor immunity. We recruited 70 hepatocellular carcinoma (HCC) patients and 30 healthy volunteers for clinical investigation, and analyzed cellular tumor immunity by using ptpro(-/-) C57BL/6 mice and NOD/SCID mice. PTPROt expression was significantly downregulated in human HCC-infiltrating T cells due to the hypoxia microenvironment; PTPROt expression highly correlated with the intratumoral Teff/Treg ratio and clinicopathologic characteristics. Moreover, PTPROt deficiency attenuated T cell-mediated anti-tumor immunity and remarkably promoted mouse HCC growth. Mechanistically, deletion of PTPROt decreased Teff quantity and quality through phosphorylation of lymphocyte-specific tyrosine kinase, but increased Treg differentiation through phosphorylation of signal transducer and activator of transcription 5. In support of the Teff/Treg homeostasis, PTPROt serves as an important tumor suppressor in HCC microenvironment. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Tyrosine Kinase Signaling in Clear Cell and Papillary Renal Cell Carcinoma Revealed by Mass Spectrometry-Based Phosphotyrosine Proteomics

PubMed Central

Haake, Scott M.; Li, Jiannong; Bai, Yun; Kinose, Fumi; Fang, Bin; Welsh, Eric; Zent, Roy; Dhillon, Jasreman; Pow-Sang, Julio; Chen, Yian Ann; Koomen, John; Rathmell, W. Kimryn; Fishman, Mayer; Haura, Eric B.

2016-01-01

Purpose Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes. Experimental Design Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. In order to describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry-based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. Results Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, while DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. Conclusions Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology amongst kidney cancer subtypes. PMID:27220961

T-cell lymphomas in South america and europe.

PubMed

Bellei, Monica; Chiattone, Carlos Sergio; Luminari, Stefano; Pesce, Emanuela Anna; Cabrera, Maria Elena; de Souza, Carmino Antonio; Gabús, Raul; Zoppegno, Lucia; Zoppegno, Lucia; Milone, Jorge; Pavlovsky, Astrid; Connors, Joseph Michael; Foss, Francine Mary; Horwitz, Steven Michael; Liang, Raymond; Montoto, Silvia; Pileri, Stefano Aldo; Polliack, Aaron; Vose, Julie Marie; Zinzani, Pier Luigi; Zucca, Emanuele; Federico, Massimo

2012-01-01

Peripheral T-cell lymphomas are a group of rare neoplasms originating from clonal proliferation of mature post-thymic lymphocytes with different entities having specific biological characteristics and clinical features. As natural killer cells are closely related to T-cells, natural killer-cell lymphomas are also part of the group. The current World Health Organization classification recognizes four categories of T/natural killer-cell lymphomas with respect to their presentation: disseminated (leukemic), nodal, extranodal and cutaneous. Geographic variations in the distribution of these diseases are well documented: nodal subtypes are more frequent in Europe and North America, while extranodal forms, including natural killer-cell lymphomas, occur almost exclusively in Asia and South America. On the whole, T-cell lymphomas are more common in Asia than in western countries, usually affect adults, with a higher tendency in men, and, excluding a few subtypes, usually have an aggressive course and poor prognosis. Apart from anaplastic lymphoma kinase-positive anaplastic large cell lymphoma, that have a good outcome, other nodal and extranodal forms have a 5-year overall survival of about 30%. According to the principal prognostic indexes, the majority of patients are allocated to the unfavorable subset. In the past, the rarity of these diseases prevented progress in the understanding of their biology and improvements in the efficaciousness of therapy. Recently, international projects devoted to these diseases created networks promoting investigations on T-cell lymphomas. These projects are the basis of forthcoming cooperative, large scale trials to detail biologic characteristics of each sub-entity and to possibly individuate targets for new therapies.

T-Cell Lymphomas in South America and Europe

PubMed Central

Bellei, Monica; Chiattone, Carlos Sergio; Luminari, Stefano; Pesce, Emanuela Anna; Cabrera, Maria Elena; de Souza, Carmino Antonio; Gabús, Raul; Zoppegno, Lucia; Zoppegno, Lucia; Milone, Jorge; Pavlovsky, Astrid; Connors, Joseph Michael; Foss, Francine Mary; Horwitz, Steven Michael; Liang, Raymond; Montoto, Silvia; Pileri, Stefano Aldo; Polliack, Aaron; Vose, Julie Marie; Zinzani, Pier Luigi; Zucca, Emanuele; Federico, Massimo

2012-01-01

Peripheral T-cell lymphomas are a group of rare neoplasms originating from clonal proliferation of mature post-thymic lymphocytes with different entities having specific biological characteristics and clinical features. As natural killer cells are closely related to T-cells, natural killer-cell lymphomas are also part of the group. The current World Health Organization classification recognizes four categories of T/natural killer-cell lymphomas with respect to their presentation: disseminated (leukemic), nodal, extranodal and cutaneous. Geographic variations in the distribution of these diseases are well documented: nodal subtypes are more frequent in Europe and North America, while extranodal forms, including natural killer-cell lymphomas, occur almost exclusively in Asia and South America. On the whole, T-cell lymphomas are more common in Asia than in western countries, usually affect adults, with a higher tendency in men, and, excluding a few subtypes, usually have an aggressive course and poor prognosis. Apart from anaplastic lymphoma kinase-positive anaplastic large cell lymphoma, that have a good outcome, other nodal and extranodal forms have a 5-year overall survival of about 30%. According to the principal prognostic indexes, the majority of patients are allocated to the unfavorable subset. In the past, the rarity of these diseases prevented progress in the understanding of their biology and improvements in the efficaciousness of therapy. Recently, international projects devoted to these diseases created networks promoting investigations on T-cell lymphomas. These projects are the basis of forthcoming cooperative, large scale trials to detail biologic characteristics of each sub-entity and to possibly individuate targets for new therapies. PMID:23049383

DNA Protecting Activities of Nymphaea nouchali (Burm. f) Flower Extract Attenuate t-BHP-Induced Oxidative Stress Cell Death through Nrf2-Mediated Induction of Heme Oxygenase-1 Expression by Activating MAP-Kinases

PubMed Central

Ju, Mi-Kyoung

2017-01-01

This study was performed to investigate the antioxidant activities of Nymphaea nouchali flower (NNF) extract and the underlying mechanism using RAW 264.7 cells. The presence of gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate, caffeic acid, quercetin, and apigenin in the NNF was confirmed by high-performance liquid chromatography (HPLC). The extract had a very potent capacity to scavenge numerous free radicals. NNF extract was also able to prevent DNA damage and quench cellular reactive oxygen species (ROS) generation induced by tert-Butyl hydroperoxide (t-BHP) with no signs of toxicity. The NNF extract was able to augment the expression of both primary and phase II detoxifying enzyme, resulting in combat the oxidative stress. This is accomplished by phosphorylation of mitogen-activated protein kinase (MAP kinase) (p38 kinase and extracellular signal-regulated kinase (ERK)) followed by enhancing the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2). This attenuates cellular ROS generation and confers protection from cell death. Altogether, the results of current study revealed that Nymphaea nouchali flower could be a source of natural phytochemicals that could lead to the development of new therapeutic agents for preventing oxidative stress associated diseases and attenuating disease progression. PMID:28956831

A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling.

PubMed

Denis, Deborah; Rouleau, Cecile; Schaffhausen, Brian S

2017-01-15

Middle T antigen (MT), the principal oncoprotein of murine polyomavirus, transforms by association with cellular proteins. Protein phosphatase 2A (PP2A), YAP, Src family tyrosine kinases, Shc, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLCγ1) have all been implicated in MT transformation. Mutant dl1015, with deletion of residues 338 to 347 in the C-terminal region, has been an enigma, because the basis for its transformation defect has not been apparent. This work probes the dl1015 region of MT. Because the region is proline rich, the hypothesis that it targets Src homology domain 3 (SH3) domains was tested, but mutation of the putative SH3 binding motif did not affect transformation. During this work, two point mutants, W348R and E349K, were identified as transformation defective. Extensive analysis of the E349K mutant is described here. Similar to wild-type MT, the E349K mutant associates with PP2A, YAP, tyrosine kinases, Shc, PI3 kinase, and PLCγ1. The E349K mutant was examined to determine the mechanism for its transformation defect. Assays of cell localization and membrane targeting showed no obvious difference in localization. Src association was normal as assayed by in vitro kinase and MT phosphopeptide mapping. Shc activation was confirmed by its tyrosine phosphorylation. Association of type 1 PI3K with MT was demonstrated by coimmunoprecipitation, showing both PI3K subunits and in vitro activity. Nonetheless, expression of the mutants failed to lead to the activation of two known downstream targets of PI3K, Akt and Rac-1. Strikingly, despite normal association of the E349K mutant with PI3K, cells expressing the mutant failed to elevate phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in mutant-expressing cells. These results indicate a novel unsuspected aspect to PI3K control. The gene coding for middle T antigen (MT) is the murine polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel

The SLP-76 SH2 domain is required for T cell development and activation

PubMed Central

Burns, Jeremy C.; Corbo, Evann; Degen, Janine; Gohil, Mercy; Anterasian, Christine; Schraven, Burkart; Koretzky, Gary A.; Kliche, Stefanie; Jordan, Martha S.

2011-01-01

The adaptor protein Src homology 2 (SH2) domain containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adaptor proteins that function together to activate second messengers required for TCR signal propagation. The N-terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with the adhesion- and degranulation-promoting adaptor protein (ADAP) and hematopoietic progenitor kinase 1 (HPK1). Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production and phosphorylation of protein kinase D (PKD) and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells. PMID:21949020

Downregulation of the c-Fes protein-tyrosine kinase inhibits the proliferation of human renal carcinoma cells

PubMed Central

Kanda, Shigeru; Miyata, Yasuyoshi; Kanetake, Hiroshi; Smithgall, Thomas E.

2009-01-01

The c-Fes protein-tyrosine kinase is associated with growth and differentiation of hematopoietic, neuronal, vascular endothelial and epithelial cell types. In this study, we investigated whether small interfering RNA (siRNA)-mediated knockdown of c-Fes expression affected proliferation of the human renal carcinoma cell lines, ACHN and VMRC-RCW. Immunofluorescence microscopy showed that c-Fes was expressed in both the cytosol and nuclei of these cells, and siRNA treatment preferentially downregulated c-Fes expression in the cytosol. Knock-down of c-Fes inhibited cellular proliferation in a dose-dependent manner with minimal increase in cell death. c-Fes siRNA treatment also downregulated the phosphorylation of Akt1 on S473 and IKKα on T23, and cyclin D1 expression, enhanced the expression of IκBα, and prevented the nuclear localization of NFκB. Treatment with an NFκB inhibitory peptide (SN50) also blocked the proliferation and nuclear localization of NFκB in these cells. The effect of SN50 treatment was not enhanced by c-Fes siRNA, suggesting that downregulation of c-Fes expression inhibited cell cycle progression through the Akt1/NFκB pathway. In contrast to siRNA-mediated knockdown, ectopic expression of either wild-type or kinase-inactive c-Fes in renal carcinoma cells failed to alter their proliferation in vitro and in vivo. Thus, suppression of proliferation resulting from siRNA-mediated knockdown may depend upon an expression of c-Fes protein rather than its kinase activity. Taken together, our results indicate that downregulation of c-Fes expression may be a potential therapeutic strategy for advanced human renal cell carcinoma and inhibition of its kinase activity as an antiangiogenic therapy does not seem to induce the growth of human renal carcinoma cells. PMID:19082481

Tangeretin inhibits extracellular-signal-regulated kinase (ERK) phosphorylation.

PubMed

Van Slambrouck, Séverine; Parmar, Virinder S; Sharma, Sunil K; De Bondt, Bart; Foré, Fleur; Coopman, Peter; Vanhoecke, Barbara W; Boterberg, Tom; Depypere, Herman T; Leclercq, Guy; Bracke, Marc E

2005-03-14

Tangeretin is a methoxyflavone from citrus fruits, which inhibits growth of human mammary cancer cells and cytolysis by natural killer cells. Attempting to unravel the flavonoid's action mechanism, we found that it inhibited extracellular-signal-regulated kinases 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent way. In human T47D mammary cancer cells this inhibition was optimally observed after priming with estradiol. The spectrum of the intracellular signalling kinase inhibition was narrow and comparison of structural congeners showed that inhibition of ERK phosphorylation was not unique for tangeretin. Our data add tangeretin to the list of small kinase inhibitors with a restricted intracellular inhibition profile.

Somatic alterations of the serine/threonine kinase LKB1 gene in squamous cell (SCC) and large cell (LCC) lung carcinoma.

PubMed

Strazisar, Mojca; Mlakar, Vid; Rott, Tomaz; Glavac, Damjan

2009-05-01

Somatic LKB1 serine/threonine kinase alterations are rare in sporadic cancers, with the exception lung adenocarcinoma, but no mutations in squamous cell or large cell primary carcinoma were discovered. We screened the LKB1 gene in 129 primary nonsmall cell lung carcinomas, adjacent healthy lung tissue, and control blood samples. Forty-five percent of nonsmall cell lung tumors harbored either intron or exon alterations. We identified R86G, F354L, Y272Y and three polymorphisms: 290+36G/T, 386+156G/T, and 862+145C/T (novel). R86G (novel) and F354L mutations were found in six squamous cell carcinomas and three large cell cancer carcinomas, but not in the adjacent healthy tissue or controls samples. The F354L mutation was found in advanced squamous cell carcinomas with elevated COX-2 expression, rare P53, and no K-RAS mutation. Results indicate that the LKB1 gene is changed in a certain proportion of nonsmall cell lung tumors, predominately in advanced squamous lung carcinoma. Inactivation of the gene takes place via the C-terminal domain and could be related to mechanisms influencing tumor initiation, differentiation, and metastasis.

Iron depletion results in Src kinase inhibition with associated cell cycle arrest in neuroblastoma cells.

PubMed

Siriwardana, Gamini; Seligman, Paul A

2015-03-01

Iron is required for cellular proliferation. Recently, using systematic time studies of neuroblastoma cell growth, we better defined the G1 arrest caused by iron chelation to a point in mid-G1, where cyclin E protein is present, but the cyclin E/CDK2 complex kinase activity is inhibited. In this study, we again used the neuroblastoma SKNSH cells lines to pinpoint the mechanism responsible for this G1 block. Initial studies showed in the presence of DFO, these cells have high levels of p27 and after reversal of iron chelation p27 is degraded allowing for CDK2 kinase activity. The initial activation of CDK2 kinase allows cells to exit G1 and enter S phase. Furthermore, we found that inhibition of p27 degradation by DFO is directly associated with inhibition of Src kinase activity measured by lack of phosphorylation of Src at the 416 residue. Activation of Src kinase occurs very early after reversal from the DFO G1 block and is temporally associated with initiation of cellular proliferation associated with entry into S phase. For the first time therefore we show that iron chelation inhibits Src kinase activity and this activity is a requirement for cellular proliferation. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

T cell specific adaptor protein (TSAd) promotes interaction of Nck with Lck and SLP-76 in T cells.

PubMed

Hem, Cecilie Dahl; Sundvold-Gjerstad, Vibeke; Granum, Stine; Koll, Lise; Abrahamsen, Greger; Buday, Laszlo; Spurkland, Anne

2015-07-11

The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated. To identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr(280) (pTyr(280)) and pTyr(305). These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr(280) and pTyr(305) on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck. TSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells.

Expression pattern and function of tyrosine receptor kinase B isoforms in rat mesenteric arterial smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Otani, Kosuke; Okada, Muneyoshi; Yamawaki, Hideyuki, E-mail: yamawaki@vmas.kitasato-u.ac.jp

Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope.more » Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells. - Highlights: • BDNF-TrkB axis mediates neurogenesis, growth, differentiation and survival. • Expression pattern and function of TrkB in vascular smooth muscle remain unclear. • Expression of TrkB FL is low, while that of TrkB T1 is the highest. • TrkB T1 contributes to maintain normal morphology possibly via activating Rho.« less

Combination of imatinib and clotrimazole enhances cell growth inhibition in T47D breast cancer cells.

PubMed

Motawi, Tarek M K; Sadik, Nermin A H; Fahim, Sally A; Shouman, Samia A

2015-05-25

Imatinib mesylate (IM), a tyrosine kinase inhibitor, is used as targeted cancer therapy. However, mono-targeting by IM does not always achieve full tumor eradication and thus it is recommended to combine IM with other anticancer agents. Clotrimazole (CLT) is an antifungal azole derivative with promising anticancer effects due to inhibiting the activity of glycolytic enzymes. The present study aimed to evaluate the effect of combining CLT with IM on breast cancer cell line in an attempt to establish effective new combination. T47D human breast cancer cell line was treated with different concentrations of IM and/or CLT for 48 h. IM-CLT interaction was determined by isobologram equation and combination index. Cell viability was confirmed by measuring LDH activity. As indicators of glycolysis inhibition, the expression of hexokinase-2 (HK-2) and 6-phosphofructo-1-kinase (PFK-1) plus the activity of intracellular lactate dehydrogenase (LDH) and pyruvate kinase (PK) were determined. In addition, glucose consumption and adenosine triphosphate (ATP) production were measured. Moreover, nitric oxide (NO), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-α (HIF-α) were also determined as they are modulators for glycolysis. This study demonstrated that IM or CLT synergistically inhibited cell growth in T47D as shown by combination and dose reduction indices. The combination of 15 μM IM and 20 μM CLT significantly decreased glucose consumption, activity of both PK and intracellular LDH, while increased leaked LDH, VEGF and NO in the medium compared to each drug alone. Furthermore the combination decreased gene expression of HK-2, PFK-1 and ATP content compared to the control. In conclusion, the synergistic effect of CLT on IM cytotoxicity in T47D cell line maybe mediated through inhibition of glycolysis and increasing both NO and VEGF. Further studies are required to confirm the efficiency and safety of this combination. Copyright © 2015 Elsevier

Anti-leukaemic activity of the TYK2 selective inhibitor NDI-031301 in T-cell acute lymphoblastic leukaemia.

PubMed

Akahane, Koshi; Li, Zhaodong; Etchin, Julia; Berezovskaya, Alla; Gjini, Evisa; Masse, Craig E; Miao, Wenyan; Rocnik, Jennifer; Kapeller, Rosana; Greenwood, Jeremy R; Tiv, Hong; Sanda, Takaomi; Weinstock, David M; Look, A Thomas

2017-04-01

Activation of tyrosine kinase 2 (TYK2) contributes to the aberrant survival of T-cell acute lymphoblastic leukaemia (T-ALL) cells. Here we demonstrate the anti-leukaemic activity of a novel TYK2 inhibitor, NDI-031301. NDI-031301 is a potent and selective inhibitor of TYK2 that induced robust growth inhibition of human T-ALL cell lines. NDI-031301 treatment of human T-ALL cell lines resulted in induction of apoptosis that was not observed with the JAK inhibitors tofacitinib and baricitinib. Further investigation revealed that NDI-031301 treatment uniquely leads to activation of three mitogen-activated protein kinases (MAPKs), resulting in phosphorylation of ERK, SAPK/JNK and p38 MAPK coincident with PARP cleavage. Activation of p38 MAPK occurred within 1 h of NDI-031301 treatment and was responsible for NDI-031301-induced T-ALL cell death, as pharmacological inhibition of p38 MAPK partially rescued apoptosis induced by TYK2 inhibitor. Finally, daily oral administration of NDI-031301 at 100 mg/kg bid to immunodeficient mice engrafted with KOPT-K1 T-ALL cells was well tolerated, and led to decreased tumour burden and a significant survival benefit. These results support selective inhibition of TYK2 as a promising potential therapeutic strategy for T-ALL. © 2017 John Wiley & Sons Ltd.

Activation of Aurora A kinase through the FGF1/FGFR signaling axis sustains the stem cell characteristics of glioblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, Yi-Chao; Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan; Kao, Chien-Yu

Fibroblast growth factor 1 (FGF1) binds and activates FGF receptors, thereby regulating cell proliferation and neurogenesis. Human FGF1 gene 1B promoter (−540 to +31)-driven SV40 T antigen has been shown to result in tumorigenesis in the brains of transgenic mice. FGF1B promoter (−540 to +31)-driven green fluorescent protein (F1BGFP) has also been used in isolating neural stem cells (NSCs) with self-renewal and multipotency from developing and adult mouse brains. In this study, we provide six lines of evidence to demonstrate that FGF1/FGFR signaling is implicated in the expression of Aurora A (AurA) and the activation of its kinase domain (Thr288more » phosphorylation) in the maintenance of glioblastoma (GBM) cells and NSCs. First, treatment of FGF1 increases AurA expression in human GBM cell lines. Second, using fluorescence-activated cell sorting, we observed that F1BGFP reporter facilitates the isolation of F1BGFP(+) GBM cells with higher expression levels of FGFR and AurA. Third, both FGFR inhibitor (SU5402) and AurA inhibitor (VX680) could down-regulate F1BGFP-dependent AurA activity. Fourth, inhibition of AurA activity by two different AurA inhibitors (VX680 and valproic acid) not only reduced neurosphere formation but also induced neuronal differentiation of F1BGFP(+) GBM cells. Fifth, flow cytometric analyses demonstrated that F1BGFP(+) GBM cells possessed different NSC cell surface markers. Finally, inhibition of AurA by VX680 reduced the neurosphere formation of different types of NSCs. Our results show that activation of AurA kinase through FGF1/FGFR signaling axis sustains the stem cell characteristics of GBM cells. Implications: This study identified a novel mechanism for the malignancy of GBM, which could be a potential therapeutic target for GBM. - Highlights: • We report that FGF1 treatment can stimulate AurA kinase expression in human GBM cells. • FGF1/FGFR signaling is involved in the activation of AurA kinase. • FGF1 sustains the self

T-kininogen inhibits kinin-mediated activation of ERK in endothelial cells.

PubMed

Leiva-Salcedo, Elias; Perez, Viviana; Acuña-Castillo, Claudio; Walter, Robin; Sierra, Felipe

2002-01-01

Serum levels of T-kininogen increase dramatically as rats approach the end of their lifespan. Stable expression of the protein in Balb/c 3T3 fibroblasts leads to a dramatic inhibition of cell proliferation, as well as inhibition of the ERK signaling pathway. T-kininogen is a potent inhibitor of cysteine proteinases, and we have described that the inhibition of ERK activity occurs, at least in part, via stabilization of the MAP kinase phosphatase, MKP-1. Since fibroblasts are not a physiological target of T-kininogen, we have now purified the protein from rat serum, and used it to assess the effect of T-kininogen on endothelial cells. Adding purified T-kininogen to EAhy 926 hybridoma cells resulted in inhibition of basal ERK activity levels, as estimated using appropriate anti-phospho ERK antibodies. Furthermore, exogenously added T-kininogen inhibited the activation of the ERK pathway induced by either bradykinin or T-kinin. We conclude that the age-related increase in hepatic T-kininogen gene expression and serum levels of the protein could have dramatic consequences on endothelial cell physiology, both under steady state conditions, and after activation by cell-specific stimuli. Our results are consistent with T-kininogen being an important modulator of the senescent phenotype in vivo.

RON kinase inhibition reduces renal endothelial injury in sickle cell disease mice

PubMed Central

Khaibullina, Alfia; Adjei, Elena A.; Afangbedji, Nowah; Ivanov, Andrey; Kumari, Namita; Almeida, Luis E.F.; Quezado, Zenaide M.N.; Nekhai, Sergei; Jerebtsova, Marina

2018-01-01

Sickle cell disease patients are at increased risk of developing a chronic kidney disease. Endothelial dysfunction and inflammation associated with hemolysis lead to vasculopathy and contribute to the development of renal disease. Here we used a Townes sickle cell disease mouse model to examine renal endothelial injury. Renal disease in Townes mice was associated with glomerular hypertrophy, capillary dilation and congestion, and significant endothelial injury. We also detected substantial renal macrophage infiltration, and accumulation of macrophage stimulating protein 1 in glomerular capillary. Treatment of human cultured macrophages with hemin or red blood cell lysates significantly increased expression of macrophage membrane-associated protease that might cleave and activate circulating macrophage stimulating protein 1 precursor. Macrophage stimulating protein 1 binds to and activates RON kinase, a cell surface receptor tyrosine kinase. In cultured human renal glomerular endothelial cells, macrophage stimulating protein 1 induced RON downstream signaling, resulting in increased phosphorylation of ERK and AKT kinases, expression of Von Willebrand factor, increased cell motility, and re-organization of F-actin. Specificity of macrophage stimulating protein 1 function was confirmed by treatment with RON kinase inhibitor BMS-777607 that significantly reduced downstream signaling. Moreover, treatment of sickle cell mice with BMS-777607 significantly reduced glomerular hypertrophy, capillary dilation and congestion, and endothelial injury. Taken together, our findings demonstrated that RON kinase is involved in the induction of renal endothelial injury in sickle cell mice. Inhibition of RON kinase activation may provide a novel approach for prevention of the development of renal disease in sickle cell disease. PMID:29519868

Tyrosine Kinase Signaling in Clear Cell and Papillary Renal Cell Carcinoma Revealed by Mass Spectrometry-Based Phosphotyrosine Proteomics.

PubMed

Haake, Scott M; Li, Jiannong; Bai, Yun; Kinose, Fumi; Fang, Bin; Welsh, Eric A; Zent, Roy; Dhillon, Jasreman; Pow-Sang, Julio M; Chen, Y Ann; Koomen, John M; Rathmell, W Kimryn; Fishman, Mayer; Haura, Eric B

2016-11-15

Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes. Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. To describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry-based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, whereas DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology among kidney cancer subtypes. Clin Cancer Res; 22(22); 5605-16. ©2016 AACR. ©2016 American Association for Cancer Research.

Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okabe, Seiichi, E-mail: okabe@tokyo-med.ac.jp; Tauchi, Tetsuzo; Tanaka, Yuko

2013-06-07

Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzedmore » the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.« less

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.

PubMed

Kutlesa, Snjezana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B; Jurecic, Roland

2009-08-01

Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.

Mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors sensitize reduced glucocorticoid response mediated by TNFalpha in human epidermal keratinocytes (HaCaT).

PubMed

Onda, Kenji; Nagashima, Masahiro; Kawakubo, Yo; Inoue, Shota; Hirano, Toshihiko; Oka, Kitaro

2006-12-08

Glucocorticoids (GCs) are essential drugs administered topically or systematically for the treatment of autoimmune skin diseases such as pemphigus. However, a certain proportion of patients does not respond well to GCs. Although studies on the relationship between cytokines and GC insensitivity in local tissues have attracted attention recently, little is known about the underlying mechanism(s) for GC insensitivity in epidermal keratinocytes. Here, we report that tumor necrosis factor (TNF) alpha reduces GC-induced transactivation of endogenous genes as well as a reporter plasmid which contains GC responsive element (GRE) in human epidermal keratinocyte cells (HaCaT). The GC insensitivity by TNFalpha was not accompanied by changes in mRNA expressions of GR isoforms (alpha or beta). However, we observed that mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors (PD98059 and U0126) significantly sensitized the GC-induced transactivation of anti-inflammatory genes (glucocorticoid-induced leucine zipper (GILZ) and mitogen-activated protein kinase phosphatase (MKP)-1) and FK506 binding protein (FKBP) 51 gene in the presence of TNFalpha. Additionally, we observed that TNFalpha reduced prednisolone (PSL)-dependent nuclear translocation of GR, which was restored by pre-treatment of MEK-1 inhibitors. This is the first study demonstrating a role of the MEK-1/ERK cascade in TNFalpha-mediated GC insensitivity. Our data suggest that overexpression of TNFalpha leads to topical GC insensitivity by reducing GR nuclear translocation in keratinocytes, and our findings also suggest that inhibiting the MEK-1/ERK cascade may offer a therapeutic potential for increasing GC efficacy in epidermis where sufficient inflammatory suppression is required.

In vitro evaluation of the Aurora kinase inhibitor VX-680 for Hepatoblastoma.

PubMed

Dewerth, Alexander; Wonner, Timo; Lieber, Justus; Ellerkamp, Verena; Warmann, Steven W; Fuchs, Jörg; Armeanu-Ebinger, Sorin

2012-06-01

Hepatoblastoma (HB) has a poor prognosis in advanced stages. The aim of this study was to enhance effectiveness of chemotherapy with antineoplastic kinase inhibitors. Viability was monitored in HB cells (HUH6, HepT1) in monolayer and spheroid cultures treated with kinase inhibitors VX-680, Wee1-InhibitorII, and SU11274 alone or in combination with cisplatin (CDDP) using MTT assays. Apoptosis was revealed by Caspase-3 assay. Western blot and immunohistochemical analyses were performed to determine histone H3 phosphorylation. Among the kinase inhibitors strongest anti-proliferative effect on HB cells was documented for VX-680. HUH6 cells responded more sensitively to the Aurora kinase inhibitor as HepT1 cells (IC(50) 8 and 16.6 μM, respectively). While VX-680 and CDDP showed no additive effects, the combination of VX-680 and histone deacetylase inhibitor SAHA had a synergistic effect on the proliferation of HUH6 cells. The inhibition with VX-680 led to reduced histone H3 phosphorylation, to an increase of apoptotic cells, and to morphological changes such as vacuolization and swelling of the cells and nuclei. The data provide evidence that VX-680 might improve treatment results in HB with increased Aurora kinase activity by inhibiting cell proliferation and induction of apoptosis.

Multi-pathway Kinase Signatures of Multipotent Stromal Cells are Predictive for Osteogenic Differentiation

PubMed Central

Platt, Manu O.; Wilder, Catera L.; Wells, Alan; Griffith, Linda G.; Lauffenburger, Douglas A.

2010-01-01

Bone marrow-derived multi-potent stromal cells (MSCs) offer great promise for regenerating tissue. While certain transcription factors have been identified in association with tendency toward particular MSC differentiation phenotypes, the regulatory network of key receptor-mediated signaling pathways activated by extracellular ligands that induce various differentiation responses remain poorly understood. Attempts to predict differentiation fate tendencies from individual pathways in isolation are problematic due to the complex pathway interactions inherent in signaling networks. Accordingly, we have undertaken a multi-variate systems approach integrating experimental measurement of multiple kinase pathway activities and osteogenic differentiation in MSCs, together with computational analysis to elucidate quantitative combinations of kinase signals predictive of cell behavior across diverse contexts. In particular, for culture on polymeric biomaterials surfaces presenting tethered epidermal growth factor (tEGF), type-I collagen, neither, or both, we have found that a partial least-squares regression model yields successful prediction of phenotypic behavior on the basis of two principal components comprising the weighted sums of 8 intracellular phosphoproteins: p-EGFR, p-Akt, p-ERK1/2, p-Hsp27, p-c-jun, p-GSK3α/β, p-p38, and p-STAT3. This combination provides strongest predictive capability for 21-day differentiated phenotype status when calculated from day-7 signal measurements (99%); day-4 (88%) and day-14 (89%) signal measurements are also significantly predictive, indicating a broad time-frame during MSC osteogenesis wherein multiple pathways and states of the kinase signaling network are quantitatively integrated to regulate gene expression, cell processes, and ultimately, cell fate. PMID:19750537

Heat shock protein 90 inhibitor NVP-AUY922 exerts potent activity against adult T-cell leukemia–lymphoma cells

PubMed Central

Taniguchi, Hiroaki; Hasegawa, Hiroo; Sasaki, Daisuke; Ando, Koji; Sawayama, Yasushi; Imanishi, Daisuke; Taguchi, Jun; Imaizumi, Yoshitaka; Hata, Tomoko; Tsukasaki, Kunihiro; Uno, Naoki; Morinaga, Yoshitomo; Yanagihara, Katsunori; Miyazaki, Yasushi

2014-01-01

Adult T-cell leukemia–lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL-related cell lines. Analysis using FACS revealed that AUY922 induced cell-cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho-Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM-1, -2, -3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922-induced death of ATL cells. In fact, SGI-1776, a pan-PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL-related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target. PMID:25263741

c-Cbl promotes T cell receptor-induced thymocyte apoptosis by activating the phosphatidylinositol 3-kinase/Akt pathway.

PubMed

Thien, Christine B F; Dagger, Samantha A; Steer, James H; Koentgen, Frank; Jansen, Elisa S; Scott, Clare L; Langdon, Wallace Y

2010-04-02

The ability of thymocytes to assess T cell receptor (TCR) signaling strength and initiate the appropriate downstream response is crucial for determining their fate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse, in which the E3 ubiquitin ligase activity of c-Cbl is inactivated, is highly sensitive to TCR-induced death signals that cause thymic deletion. This high intensity signal involves the enhanced tyrosine phosphorylation of the mutant c-Cbl protein promoting a marked increase in the activation of Akt. Here we show that this high intensity signal in c-Cbl RING finger mutant thymocytes also promotes the enhanced induction of two mediators of TCR-directed thymocyte apoptosis, Nur77 and the pro-apoptotic Bcl-2 family member, Bim. In contrast, a knock-in mouse harboring a mutation at Tyr-737, the site in c-Cbl that activates phosphatidylinositol 3-kinase, shows reduced TCR-mediated responses including suppression of Akt activation, a reduced induction of Nur77 and Bim, and greater resistance to thymocyte death. These findings identify tyrosine-phosphorylated c-Cbl as a critical sensor of TCR signal strength that regulates the engagement of death-promoting signals.

N-ras couples antigen receptor signaling to Eomesodermin and to functional CD8+ T cell memory but not to effector differentiation

PubMed Central

Iborra, Salvador; Ramos, Manuel; Arana, David M.; Lázaro, Silvia; Aguilar, Francisco; Santos, Eugenio; López, Daniel

2013-01-01

Signals from the TCR that specifically contribute to effector versus memory CD8+ T cell differentiation are poorly understood. Using mice and adoptively transferred T lymphocytes lacking the small GTPase N-ras, we found that N-ras–deficient CD8+ T cells differentiate efficiently into antiviral primary effectors but have a severe defect in generating protective memory cells. This defect was rescued, although only partly, by rapamycin-mediated inhibition of mammalian target of rapamycin (mTOR) in vivo. The memory defect correlated with a marked impairment in vitro and in vivo of the antigen-mediated early induction of T-box transcription factor Eomesodermin (Eomes), whereas T-bet was unaffected. Besides N-ras, early Eomes induction in vitro required phosphoinositide 3-kinase (PI3K)–AKT but not extracellular signal-regulated kinase (ERK) activation, and it was largely insensitive to rapamycin. Consistent with N-ras coupling Eomes to T cell memory, retrovirally enforced expression of Eomes in N-ras–deficient CD8+ T cells effectively rescued their memory differentiation. Thus, our study identifies a critical role for N-ras as a TCR-proximal regulator of Eomes for early determination of the CD8+ T cell memory fate. PMID:23776078

The hydroxyflavone, fisetin, suppresses mast cell activation induced by interaction with activated T cell membranes

PubMed Central

Nagai, K; Takahashi, Y; Mikami, I; Fukusima, T; Oike, H; Kobori, M

2009-01-01

Background and purpose: Cell-to-cell interactions between mast cells and activated T cells are increasingly recognized as a possible mechanism in the aetiology of allergic or non-allergic inflammatory disorders. To determine the anti-allergic effect of fisetin, we examined the ability of fisetin to suppress activation of the human mast cell line, HMC-1, induced by activated Jurkat T cell membranes. Experimental approach: HMC-1 cells were incubated with or without fisetin for 15 min and then co-cultured with Jurkat T cell membranes activated by phorbol-12-myristate 13-acetate for 16 h. We determined gene expression in activated HMC-1 cells by DNA microarray and quantitative reverse transcription (RT)-PCR analysis. We also examined activation of the transcription factor NF-κB and MAP kinases (MAPKs) in activated HMC-1 cells. Key results: Fisetin suppresses cell spreading and gene expression in HMC-1 cells stimulated by activated T cell membranes. Additionally, we show that these stimulated HMC-1 cells expressed granzyme B. The stimulatory interaction also induced activation of NF-κB and MAPKs; these activations were suppressed by fisetin. Fisetin also reduced the amount of cell surface antigen CD40 and intercellular adhesion molecule-1 (ICAM-1) on activated HMC-1 cells. Conclusions and implications: Fisetin suppressed activation of HMC-1 cells by activated T cell membranes by interfering with cell-to-cell interaction and inhibiting the activity of NF-κB and MAPKs and thereby suppressing gene expression. Fisetin may protect against the progression of inflammatory diseases by limiting interactions between mast cells and activated T cells. PMID:19702784

Engineering Chimeric Antigen Receptor T cells to Treat Glioblastoma.

PubMed

Choi, Bryan D; O'Rourke, Donald M; Maus, Marcela V

2017-08-01

Immunotherapy has emerged as a promising strategy for glioblastoma (GBM), a disease that remains universally fatal despite currently available standard-of-care. Adoptive T cell therapy has been shown to produce potent antitumor immunity while obviating the need for traditional antigen presentation and primary immune responses. Chimeric antigen receptors (CARs) are specialized molecules that can be expressed on the surface of T cells allowing for redirected cytotoxicity against tumor antigens of interest. To date, the application of CAR T cells for GBM has been relatively limited, in large part due to a dearth of well-described tumor specific antigens that are both homogenously and frequently expressed. A mutated version of the epidermal growth factor receptor, EGFRvIII, is a constitutively activated tyrosine kinase that is expressed on the surface of GBM and other common neoplasms, but completely absent from all normal tissues. We have recently generated CAR T cells directed against EGFRvIII and reported results from a Phase I clinical trial investigating this platform in patients with EGFRvIII-expressing GBM. Our study showed that despite conventional notions of central nervous system "immune-privilege," EGFRvIII CAR T cells trafficked to intracerebral tumors, leading to successful targeting and eradication of this antigen in the brain. Here, we review our experience with EGFRvIII CAR T cells and highlight important considerations for the clinical translation of this therapy in patients with GBM.

c-Abl-Mediated Tyrosine Phosphorylation of the T-bet DNA-Binding Domain Regulates CD4+ T-Cell Differentiation and Allergic Lung Inflammation ▿

PubMed Central

Chen, An; Lee, Sang-Myeong; Gao, Beixue; Shannon, Stephen; Zhu, Zhou; Fang, Deyu

2011-01-01

The tyrosine kinase c-Abl is required for full activation of T cells, while its role in T-cell differentiation has not been characterized. We report that c-Abl deficiency skews CD4+ T cells to type 2 helper T cell (Th2) differentiation, and c-Abl−/− mice are more susceptible to allergic lung inflammation. c-Abl interacts with and phosphorylates T-bet, a Th1 lineage transcription factor. c-Abl-mediated phosphorylation enhances the transcriptional activation of T-bet. Interestingly, three tyrosine residues within the T-bet DNA-binding domain are the predominant sites of phosphorylation by c-Abl. Mutation of these tyrosine residues inhibits the promoter DNA-binding activity of T-bet. c-Abl regulates Th cell differentiation in a T-bet-dependent manner because genetic deletion of T-bet in CD4+ T cells abolishes c-Abl-deficiency-mediated enhancement of Th2 differentiation. Reintroduction of T-bet-null CD4+ T cells with wild-type T-bet, but not its tyrosine mutant, rescues gamma interferon (IFN-γ) production and inhibits Th2 cytokine production. Therefore, c-Abl catalyzes tyrosine phosphorylation of the DNA-binding domain of T-bet to regulate CD4+ T cell differentiation. PMID:21690296

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, Harry A.; Gadbois, Donna M.; Tobey, Robert A.; Bradbury, E. Morton

1993-01-01

A G.sub.1 phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G.sub.1 phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G.sub.1 cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G.sub.1 phase, suggesting that such G.sub.1 phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, H.A.; Gadbois, D.M.; Tobey, R.A.; Bradbury, E.M.

1993-02-09

A G[sub 1] phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G[sub 1] phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G[sub 1] cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G[sub 1] phase, suggesting that such G[sub 1] phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

A Tumor Cell-Selective Inhibitor of Mitogen-Activated Protein Kinase Phosphatases Sensitizes Breast Cancer Cells to Lymphokine-Activated Killer Cell Activity

PubMed Central

Kaltenmeier, Christof T.; Vollmer, Laura L.; Vernetti, Lawrence A.; Caprio, Lindsay; Davis, Keanu; Korotchenko, Vasiliy N.; Day, Billy W.; Tsang, Michael; Hulkower, Keren I.; Lotze, Michael T.

2017-01-01

Dual specificity mitogen-activated protein kinase (MAPK) phosphatases [dual specificity phosphatase/MAP kinase phosphatase (DUSP-MKP)] have been hypothesized to maintain cancer cell survival by buffering excessive MAPK signaling caused by upstream activating oncogenic products. A large and diverse body of literature suggests that genetic depletion of DUSP-MKPs can reduce tumorigenicity, suggesting that hyperactivating MAPK signaling by DUSP-MKP inhibitors could be a novel strategy to selectively affect the transformed phenotype. Through in vivo structure-activity relationship studies in transgenic zebrafish we recently identified a hyperactivator of fibroblast growth factor signaling [(E)-2-benzylidene-5-bromo-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI-215)] that is devoid of developmental toxicity and restores defective MAPK activity caused by overexpression of DUSP1 and DUSP6 in mammalian cells. Here, we hypothesized that BCI-215 could selectively affect survival of transformed cells. In MDA-MB-231 human breast cancer cells, BCI-215 inhibited cell motility, caused apoptosis but not primary necrosis, and sensitized cells to lymphokine-activated killer cell activity. Mechanistically, BCI-215 induced rapid and sustained phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) in the absence of reactive oxygen species, and its toxicity was partially rescued by inhibition of p38 but not JNK or ERK. BCI-215 also hyperactivated MKK4/SEK1, suggesting activation of stress responses. Kinase phosphorylation profiling documented BCI-215 selectively activated MAPKs and their downstream substrates, but not receptor tyrosine kinases, SRC family kinases, AKT, mTOR, or DNA damage pathways. Our findings support the hypothesis that BCI-215 causes selective cancer cell cytotoxicity in part through non-redox-mediated activation of MAPK signaling, and the findings also identify an intersection with immune cell killing that is

Targeting the T-Lak cell originated protein kinase by OTS964 shrinks the size of power-law coded heterogeneous glioma stem cell populations

PubMed Central

Sugimori, Michiya; Hayakawa, Yumiko; Koh, Masaki; Hayashi, Tomohide; Tamura, Ryoi; Kuroda, Satoshi

2018-01-01

Glioblastoma resists chemoradiotherapy, then, recurs to be a fatal space-occupying lesion. The recurrence is caused by re-growing cell populations such as glioma stem cells (GSCs), suggesting that GSC populations should be targeted. This study addressed whether a novel anti-cancer drug, OTS964, an inhibitor for T-LAK cell originated protein kinase (TOPK), is effective in reducing the size of the heterogeneous GSC populations, a power-law coded heterogeneous GSC populations consisting of glioma sphere (GS) clones, by detailing quantitative growth properties. We found that OTS964 killed GS clones while suppressing the growth of surviving GS clones, thus identifying clone-eliminating and growth-disturbing efficacies of OTS964. The efficacies led to a significant size reduction in GS populations in a dose-dependent manner. The surviving GS clones reconstructed GS populations in the following generations; the recovery of GS populations fits a recurrence after the chemotherapy. The recovering GS clones resisted the clone-eliminating effect of OTS964 in sequential exposure during the growth recovery. However, surprisingly, the resistant properties of the recovered-GS clones had been plastically canceled during self-renewal, and then the GS clones had become re-sensitive to OTS964. Thus, OTS964 targets GSCs to eliminate them or suppress their growth, resulting in shrinkage of the power-law coded GSC populations. We propose a therapy focusing on long-term control in recurrence of glioblastoma via reducing the size of the GSC populations by OTS964. PMID:29423027

Targeting the T-Lak cell originated protein kinase by OTS964 shrinks the size of power-law coded heterogeneous glioma stem cell populations.

PubMed

Sugimori, Michiya; Hayakawa, Yumiko; Koh, Masaki; Hayashi, Tomohide; Tamura, Ryoi; Kuroda, Satoshi

2018-01-09

Glioblastoma resists chemoradiotherapy, then, recurs to be a fatal space-occupying lesion. The recurrence is caused by re-growing cell populations such as glioma stem cells (GSCs), suggesting that GSC populations should be targeted. This study addressed whether a novel anti-cancer drug, OTS964, an inhibitor for T-LAK cell originated protein kinase (TOPK), is effective in reducing the size of the heterogeneous GSC populations, a power-law coded heterogeneous GSC populations consisting of glioma sphere (GS) clones, by detailing quantitative growth properties. We found that OTS964 killed GS clones while suppressing the growth of surviving GS clones, thus identifying clone-eliminating and growth-disturbing efficacies of OTS964. The efficacies led to a significant size reduction in GS populations in a dose-dependent manner. The surviving GS clones reconstructed GS populations in the following generations; the recovery of GS populations fits a recurrence after the chemotherapy. The recovering GS clones resisted the clone-eliminating effect of OTS964 in sequential exposure during the growth recovery. However, surprisingly, the resistant properties of the recovered-GS clones had been plastically canceled during self-renewal, and then the GS clones had become re-sensitive to OTS964. Thus, OTS964 targets GSCs to eliminate them or suppress their growth, resulting in shrinkage of the power-law coded GSC populations. We propose a therapy focusing on long-term control in recurrence of glioblastoma via reducing the size of the GSC populations by OTS964.

p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

PubMed

Cereseto, A; Diella, F; Mulloy, J C; Cara, A; Michieli, P; Grassmann, R; Franchini, G; Klotman, M E

1996-09-01

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

N-acetylcysteine attenuates TNF-α-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells

PubMed Central

Hashimoto, Shu; Gon, Yasuhiro; Matsumoto, Ken; Takeshita, Ikuko; Horie, Takashi

2001-01-01

We have previously shown that tumour necrosis factor-α (TNF-α) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H2O2 generated by TNF-α can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-α-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-α and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. Intracellular GSH levels increased in NAC-treated cells. NAC attenuated TNF-α-induced activation of p38 MAP kinase and MKK3/MKK6. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-α-stimulated cells. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury. PMID:11156586

N-acetylcysteine attenuates TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells.

PubMed

Hashimoto, S; Gon, Y; Matsumoto, K; Takeshita, I; Horie, T

2001-01-01

1. We have previously shown that tumour necrosis factor-alpha (TNF-alpha) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H(2)O(2) generated by TNF-alpha can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-alpha-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. 2. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-alpha and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. 3. Intracellular GSH levels increased in NAC-treated cells. 4. NAC attenuated TNF-alpha-induced activation of p38 MAP kinase and MKK3/MKK6. 5. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-alpha-stimulated cells. 6. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury.

Dysfunctional BLK in common variable immunodeficiency perturbs B-cell proliferation and ability to elicit antigen-specific CD4+ T-cell help.

PubMed

Compeer, Ewoud B; Janssen, Willemijn; van Royen-Kerkhof, Annet; van Gijn, Marielle; van Montfrans, Joris M; Boes, Marianne

2015-05-10

Common Variable Immunodeficiency (CVID) is the most prevalent primary antibody deficiency, and characterized by defective generation of high-affinity antibodies. Patients have therefore increased risk to recurrent infections of the respiratory and intestinal tract. Development of high-affinity antigen-specific antibodies involves two key actions of B-cell receptors (BCR): transmembrane signaling through BCR-complexes to induce B-cell differentiation and proliferation, and BCR-mediated antigen internalization for class-II MHC-mediated presentation to acquire antigen-specific CD4(+) T-cell help.We identified a variant (L3P) in the B-lymphoid tyrosine kinase (BLK) gene of 2 related CVID-patients, which was absent in healthy relatives. BLK belongs to the Src-kinases family and involved in BCR-signaling. Here, we sought to clarify BLK function in healthy human B-cells and its association to CVID.BLK expression was comparable in patient and healthy B-cells. Functional analysis of L3P-BLK showed reduced BCR crosslinking-induced Syk phosphorylation and proliferation, in both primary B-cells and B-LCLs. B-cells expressing L3P-BLK showed accelerated destruction of BCR-internalized antigen and reduced ability to elicit CD40L-expression on antigen-specific CD4(+) T-cells.In conclusion, we found a novel BLK gene variant in CVID-patients that causes suppressed B-cell proliferation and reduced ability of B-cells to elicit antigen-specific CD4(+) T-cell responses. Both these mechanisms may contribute to hypogammaglobulinemia in CVID-patients.

Breast implant-associated, ALK-negative, T-cell, anaplastic, large-cell lymphoma: establishment and characterization of a model cell line (TLBR-1) for this newly emerging clinical entity.

PubMed

Lechner, Melissa G; Lade, Stephen; Liebertz, Daniel J; Prince, H Miles; Brody, Garry S; Webster, Howard R; Epstein, Alan L

2011-04-01

Primary lymphomas of the breast are very rare (0.2-1.5% of breast malignancies) and the vast majority (95%) are of B-cell origin. Recently, 40 cases of clinically indolent anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide. A tumor biopsy specimen from a patient in this series was obtained for characterization. By using a human stromal feeder layer and IL-2, a novel cell line, TLBR-1, was established from this biopsy and investigated by using cytogenetics and various biomolecular methods. Immunoperoxidase staining of the tumor biopsy showed a CD30/CD8/CD4 coexpressing T-cell population that was epithelial membrane antigen (EMA)(+) and perforin(+) . Multiplex polymerase chain reaction (PCR) of TCRγ genes showed monoclonality that suggested a T-cell origin, yet pan-T markers CD2/5/7, anaplastic large-cell kinase (ALK)-1, pancytokeratins, CD20, CD56, and Epstein-Barr virus (EBV) by in situ hybridization (ISH) were negative. TLBR-1 is IL-2 dependent, has a relatively long doubling time (55 hours), and displays different cellular shapes in culture. Cytogenetic analysis of tumor and TLBR-1 cells confirmed a highly anaplastic cell population with a modal number of 47 chromosomes lacking t(2;5). PCR screens for EBV and human T-lymphotropic virus types 1 and 2 (HTLV-1/2) were negative. Fluorescence-activated cell-sorting (FACS) analysis showed strong positivity for CD4/8, CD30, CD71, and CD26 expression, and antigen presentation (HLA-DR(+) CD80(+) CD86(+) ), IL-2 signaling (CD25(+) CD122(+) ), and NK (CD56(+) ) markers, and Western blots demonstrated strong Notch1 expression. Severe combined immunodeficiency (SCID) mouse TLBR-1 heterotransplants recapitulated the histology and marker characteristics of the original tumor. TLBR-1, a novel ALK-negative, T-cell, anaplastic, large-cell lymphoma, closely resembles the original biopsy and represents an important tool for studying this newly recognized

Breast Implant-Associated, ALK-Negative, T-Cell, Anaplastic, Large-Cell Lymphoma: Establishment and Characterization of a Model Cell Line (TLBR-1) for This Newly Emerging Clinical Entity

PubMed Central

Lechner, Melissa G.; Lade, Stephen; Liebertz, Daniel J.; Prince, H. Miles; Brody, Garry S.; Webster, Howard R.; Epstein, Alan L.

2014-01-01

BACKGROUND Primary lymphomas of the breast are very rare (0.2–1.5% of breast malignancies) and the vast majority (95%) are of B-cell origin. Recently, 40 cases of clinically indolent anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide. METHODS A tumor biopsy specimen from a patient in this series was obtained for characterization. By using a human stromal feeder layer and IL-2, a novel cell line, TLBR-1, was established from this biopsy and investigated by using cytogenetics and various biomolecular methods. RESULTS Immunoperoxidase staining of the tumor biopsy showed a CD30/CD8/CD4 coexpressing T-cell population that was epithelial membrane antigen (EMA)+ and perforin+. Multiplex polymerase chain reaction (PCR) of TCRγ genes showed monoclonality that suggested a T-cell origin, yet pan-T markers CD2/5/7, anaplastic large-cell kinase (ALK)-1, pancytokeratins, CD20, CD56, and Epstein-Barr virus (EBV) by in situ hybridization (ISH) were negative. TLBR-1 is IL-2 dependent, has a relatively long doubling time (55 hours), and displays different cellular shapes in culture. Cytogenetic analysis of tumor and TLBR-1 cells confirmed a highly anaplastic cell population with a modal number of 47 chromosomes lacking t(2;5). PCR screens for EBV and human T-lymphotropic virus types 1 and 2 (HTLV-1/2) were negative. Fluorescence-activated cell-sorting (FACS) analysis showed strong positivity for CD4/8, CD30, CD71, and CD26 expression, and antigen presentation (HLA-DR+CD80+CD86+), IL-2 signaling (CD25+CD122+), and NK (CD56+) markers, and Western blots demonstrated strong Notch1 expression. Severe combined immunodeficiency (SCID) mouse TLBR-1 heterotransplants recapitulated the histology and marker characteristics of the original tumor. CONCLUSIONS TLBR-1, a novel ALK-negative, T-cell, anaplastic, large-cell lymphoma, closely resembles the original biopsy and represents an important tool for studying this

Maternal Embryonic Leucine-zipper Kinase: Key Kinase for Stem Cell Phenotype in Glioma and Other Cancers

PubMed Central

Ganguly, Ranjit; Hong, Christopher; Smith, Luke; Kornblum, Harley I; Nakano, Ichiro

2014-01-01

Maternal embryonic leucine zipper kinase (MELK) is a member of the snf1/AMPK family of protein Serine/Threonine kinases that has recently gained significant attention in the stem cell and cancer biology field. Recent studies suggest that activation of this kinase is tightly associated with extended survival and accelerated proliferation of cancer stem cells (CSCs) in various organs. Overexpression of MELK has been noted in various cancers, including colon, breast, ovaries, pancreas, prostate, and brain, making the inhibition of MELK an attractive therapeutic strategy for a variety of cancers. In the experimental cancer models, depletion of MELK by RNA interference or small molecule inhibitors induces apoptotic cell death of cancer stem cells derived from glioblastoma and breast cancer, both in vitro and in vivo. Mechanism of action of MELK includes, yet may not be restricted to, direct binding and activation of the oncogenic transcription factors c-JUN and FOXM1 in cancer cells but not in the normal counterparts. Following these pre-clinical studies, the Phase I clinical trial for advanced cancers with OTS167 started in 2013, as the first-in-class MELK inhibitor. This review summarizes the current molecular understanding of MELK and the recent pre-clinical studies about MELK as a cancer therapeutic target. PMID:24795222

The discovery of a reciprocal relationship between tyrosine-kinase signaling and cullin neddylation.

PubMed

Friend, Samantha F; Peterson, Lisa K; Treacy, Eric; Stefanski, Adrianne L; Sosinowski, Tomasz; Pennock, Nathan D; Berger, Allison J; Winn, Virginia D; Dragone, Leonard L

2013-01-01

While neddylation is known to activate cullin (CUL)-RING ubiquitin ligases (CRLs), its role in regulating T cell signaling is poorly understood. Using the investigational NEDD8 activating enzyme (NAE) inhibitor, MLN4924, we found that neddylation negatively regulates T cell receptor (TCR) signaling, as its inhibition increases IL-2 production, T cell proliferation and Treg development in vitro. We also discovered that loss of CUL neddylation occurs upon TCR signaling, and CRLs negatively regulate IL-2 production. Additionally, we found that tyrosine kinase signaling leads to CUL deneddylation in multiple cell types. These studies indicate that CUL neddylation is a global regulatory mechanism for tyrosine kinase signaling.

T-LAK Cell-Originated Protein Kinase (TOPK) as a Prognostic Factor and a Potential Therapeutic Target in Ovarian Cancer.

PubMed

Ikeda, Yuji; Park, Jae-Hyun; Miyamoto, Takashi; Takamatsu, Naofumi; Kato, Taigo; Iwasa, Akiko; Okabe, Shuhei; Imai, Yuichi; Fujiwara, Keiichi; Nakamura, Yusuke; Hasegawa, Kosei

2016-12-15

We aimed to clarify the clinical significance of TOPK (T-lymphokine-activated killer cell-originated protein kinase) expression in ovarian cancer and evaluate the possible effect of TOPK inhibitors, OTS514 and OTS964, on ovarian cancer cells. TOPK expression was examined by immunohistochemistry using 163 samples with epithelial ovarian cancer (EOC). TOPK protein level and FOXM1 transcriptional level in ovarian cancer cell lines were examined by Western blot and RT-PCR, respectively. Half-maximum inhibitory concentration (IC 50 ) values against TOPK inhibitors were examined by the MTT assay. Using the peritoneal dissemination model of ES-2 ovarian cancer cells, we examined the in vivo efficacy of OTS514. In addition, the cytotoxic effect of OTS514 and OTS964 on 31 patient-derived primary ovarian cancer cells was examined. TOPK was expressed very highly in 84 (52%) of 163 EOC tissues, and high TOPK expression was significantly associated with poor progression-free survival and overall survival in early-stage cases of EOC (P = 0.008 and 0.006, respectively). Both OTS514 and OTS964 showed significant growth-inhibitory effect on ovarian cancer cell lines with IC 50 values of 3.0 to 46 nmol/L and 14 to 110 nmol/L, respectively. TOPK protein and transcriptional levels of FOXM1 were reduced by TOPK inhibitor treatment. Oral administration of OTS514 significantly elongated overall survival in the ES-2 abdominal dissemination xenograft model, compared with vehicle control (P < 0.001). Two drugs showed strong growth-inhibitory effect on primary ovarian cancer cells regardless of tumor sites or histological subtypes. Our results demonstrated the clinical significance of high TOPK expression and potential of TOPK inhibitors to treat ovarian cancer. Clin Cancer Res; 22(24); 6110-7. ©2016 AACR. ©2016 American Association for Cancer Research.

Mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors sensitize reduced glucocorticoid response mediated by TNF{alpha} in human epidermal keratinocytes (HaCaT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Onda, Kenji; Nagashima, Masahiro; Kawakubo, Yo

2006-12-08

Glucocorticoids (GCs) are essential drugs administered topically or systematically for the treatment of autoimmune skin diseases such as pemphigus. However, a certain proportion of patients does not respond well to GCs. Although studies on the relationship between cytokines and GC insensitivity in local tissues have attracted attention recently, little is known about the underlying mechanism(s) for GC insensitivity in epidermal keratinocytes. Here, we report that tumor necrosis factor (TNF) {alpha} reduces GC-induced transactivation of endogenous genes as well as a reporter plasmid which contains GC responsive element (GRE) in human epidermal keratinocyte cells (HaCaT). The GC insensitivity by TNF{alpha} wasmore » not accompanied by changes in mRNA expressions of GR isoforms ({alpha} or {beta}). However, we observed that mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors (PD98059 and U0126) significantly sensitized the GC-induced transactivation of anti-inflammatory genes (glucocorticoid-induced leucine zipper (GILZ) and mitogen-activated protein kinase phosphatase (MKP)-1) and FK506 binding protein (FKBP) 51 gene in the presence of TNF{alpha}. Additionally, we observed that TNF{alpha} reduced prednisolone (PSL)-dependent nuclear translocation of GR, which was restored by pre-treatment of MEK-1 inhibitors. This is the first study demonstrating a role of the MEK-1/ERK cascade in TNF{alpha}-mediated GC insensitivity. Our data suggest that overexpression of TNF{alpha} leads to topical GC insensitivity by reducing GR nuclear translocation in keratinocytes, and our findings also suggest that inhibiting the MEK-1/ERK cascade may offer a therapeutic potential for increasing GC efficacy in epidermis where sufficient inflammatory suppression is required.« less

Mutant JAK3 phosphoproteomic profiling predicts synergism between JAK3 inhibitors and MEK/BCL2 inhibitors for the treatment of T-cell acute lymphoblastic leukemia

PubMed Central

Degryse, S; de Bock, C E; Demeyer, S; Govaerts, I; Bornschein, S; Verbeke, D; Jacobs, K; Binos, S; Skerrett-Byrne, D A; Murray, H C; Verrills, N M; Van Vlierberghe, P; Cools, J; Dun, M D

2018-01-01

Mutations in the interleukin-7 receptor (IL7R) or the Janus kinase 3 (JAK3) kinase occur frequently in T-cell acute lymphoblastic leukemia (T-ALL) and both are able to drive cellular transformation and the development of T-ALL in mouse models. However, the signal transduction pathways downstream of JAK3 mutations remain poorly characterized. Here we describe the phosphoproteome downstream of the JAK3(L857Q)/(M511I) activating mutations in transformed Ba/F3 lymphocyte cells. Signaling pathways regulated by JAK3 mutants were assessed following acute inhibition of JAK1/JAK3 using the JAK kinase inhibitors ruxolitinib or tofacitinib. Comprehensive network interrogation using the phosphoproteomic signatures identified significant changes in pathways regulating cell cycle, translation initiation, mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signaling, RNA metabolism, as well as epigenetic and apoptotic processes. Key regulatory proteins within pathways that showed altered phosphorylation following JAK inhibition were targeted using selumetinib and trametinib (MEK), buparlisib (PI3K) and ABT-199 (BCL2), and found to be synergistic in combination with JAK kinase inhibitors in primary T-ALL samples harboring JAK3 mutations. These data provide the first detailed molecular characterization of the downstream signaling pathways regulated by JAK3 mutations and provide further understanding into the oncogenic processes regulated by constitutive kinase activation aiding in the development of improved combinatorial treatment regimens. PMID:28852199

PKCθ links proximal T cell and Notch signaling through localized regulation of the actin cytoskeleton

PubMed Central

Britton, Graham J; Ambler, Rachel; Clark, Danielle J; Hill, Elaine V; Tunbridge, Helen M; McNally, Kerrie E; Burton, Bronwen R; Butterweck, Philomena; Sabatos-Peyton, Catherine; Hampton-O’Neil, Lea A; Verkade, Paul; Wülfing, Christoph; Wraith, David Cameron

2017-01-01

Notch is a critical regulator of T cell differentiation and is activated through proteolytic cleavage in response to ligand engagement. Using murine myelin-reactive CD4 T cells, we demonstrate that proximal T cell signaling modulates Notch activation by a spatiotemporally constrained mechanism. The protein kinase PKCθ is a critical mediator of signaling by the T cell antigen receptor and the principal costimulatory receptor CD28. PKCθ selectively inactivates the negative regulator of F-actin generation, Coronin 1A, at the center of the T cell interface with the antigen presenting cell (APC). This allows for effective generation of the large actin-based lamellum required for recruitment of the Notch-processing membrane metalloproteinase ADAM10. Such enhancement of Notch activation is critical for efficient T cell proliferation and Th17 differentiation. We reveal a novel mechanism that, through modulation of the cytoskeleton, controls Notch activation at the T cell:APC interface thereby linking T cell receptor and Notch signaling pathways. DOI: http://dx.doi.org/10.7554/eLife.20003.001 PMID:28112644

Effects of Butyltins (BTs) on Mitogen-Activated-Protein Kinase Kinase Kinase (MAP3K) and Ras Activity in Human Natural Killer Cells

PubMed Central

Celada, Lindsay J.; Whalen, Margaret M.

2013-01-01

Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT), diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally-infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to the activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 minutes of TBT exposure and the MAP3K, ASK1, after one hour exposures to TBT. In addition, our results suggest that both TBT and DBT are impacting the regulation of c-Raf. PMID:24038145

Heat shock protein 90 inhibitor NVP-AUY922 exerts potent activity against adult T-cell leukemia-lymphoma cells.

PubMed

Taniguchi, Hiroaki; Hasegawa, Hiroo; Sasaki, Daisuke; Ando, Koji; Sawayama, Yasushi; Imanishi, Daisuke; Taguchi, Jun; Imaizumi, Yoshitaka; Hata, Tomoko; Tsukasaki, Kunihiro; Uno, Naoki; Morinaga, Yoshitomo; Yanagihara, Katsunori; Miyazaki, Yasushi

2014-12-01

Adult T-cell leukemia-lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL-related cell lines. Analysis using FACS revealed that AUY922 induced cell-cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho-Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM-1, -2, -3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922-induced death of ATL cells. In fact, SGI-1776, a pan-PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL-related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.