RESUMO
T-cell exhaustion was initially identified in chronic infection in mice and was subsequently described in humans with cancer. Although the distinct signature of exhausted T (TEX) cells in cancer has been well investigated, the molecular mechanism of T-cell exhaustion in cancer is not fully understood. Using single-cell RNA sequencing, we report here that TEX cells in esophageal cancer are more heterogeneous than previously clarified. Sprouty RTK signaling antagonist 1 (SPRY1) was notably enriched in two subsets of exhausted CD8+ T cells. When overexpressed, SPRY1 impaired T-cell activation by interacting with CBL, a negative regulator of ZAP-70 tyrosine phosphorylation. Data from the Tumor Immune Estimation Resource revealed a strong correlation between FGF2 and SPRY1 expression in esophageal cancer. High expression of FGF2 was evident in fibroblasts from esophageal cancer tissue and correlated with poor overall survival. In vitro administration of FGF2 significantly upregulated expression of SPRY1 in CD8+ T cells and attenuated T-cell receptor-triggered CD8+ T-cell activation. A mouse tumor model confirmed that overexpression of FGF2 in fibroblasts significantly upregulated SPRY1 expression in TEX cells, impaired T-cell cytotoxic activity, and promoted tumor growth. Thus, these findings identify FGF2 as an important regulator of SPRY1 expression involved in establishing the dysfunctional state of CD8+ T cells in esophageal cancer. SIGNIFICANCE: These findings reveal FGF2 as an important regulator of SPRY1 expression involved in establishing the dysfunctional state of CD8+ T cells and suggest that inhibition of FGF2 has potential clinical value in ESCC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5583/F1.large.jpg.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos T CD8-Positivos/imunologia , Fibroblastos Associados a Câncer/metabolismo , Neoplasias Esofágicas/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Animais , Modelos Animais de Doenças , Neoplasias Esofágicas/patologia , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Células Jurkat , Ativação Linfocitária , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína Oncogênica v-cbl/genética , Proteína Oncogênica v-cbl/metabolismo , Fosfoproteínas/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Juvenile myelomonocytic leukaemia (JMML) is a rare clonal disorder of early childhood. Constitutive activation of the RAS pathway is the initial event in JMML. Around 90% of patients diagnosed with JMML carry a mutation in the PTPN11, NRAS, KRAS, NF1 or CBL genes. It has been demonstrated that after this first genetic event, an additional somatic mutation or epigenetic modification is involved in disease progression. The available genetic and clinical data have enabled researchers to establish relationships between JMML and several clinical conditions, including Noonan syndrome, Ras-associated lymphoproliferative disease, and Moyamoya disease. Despite scientific progress and the development of more effective treatments, JMML is still a deadly disease: the 5-year survival rate is ~50%. Here, we report on recent research having led to a better understanding of the genetic and molecular mechanisms involved in JMML.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mielomonocítica Juvenil/genética , Mutação , Animais , Epigênese Genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Leucemia Mielomonocítica Juvenil/metabolismo , Leucemia Mielomonocítica Juvenil/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Proteína Oncogênica v-cbl/genética , Proteína Oncogênica v-cbl/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
OBJECTIVE: To investigate the effect of ascorbic acid (AA) on hemostatic function during living donor liver transplantation (LDLT). METHODS: Blood samples from 21 LDLT recipients were taken within 30 minutes after induction and at 120 minutes after reperfusion. Rotational thromboelastography (TEG) and western blot analysis were used to analyze for fibrinolysis and functional changes in c-Cbl and Cbl-b, respectively. TEG test samples were prepared as one of three groups: C group (0.36 mL of blood), N group (0.324 mL of blood + 0.036 mL of 0.9% normal saline), and A group (0.324 mL of blood + 0.036 mL of 200 µmol/L-AA dissolved in 0.9% normal saline). RESULTS: AA decreased fibrinolysis and increased clot rigidity at baseline and 120 minutes after reperfusion. Cbl-b expression was significantly increased at baseline and 120 minutes after reperfusion in the A group compared with the C and N groups. However, c-Cbl phosphorylation was most significantly decreased in the A group at baseline and 120 minutes after reperfusion. CONCLUSION: AA can significantly decrease fibrinolysis and improve clot rigidity in LT recipients during LDLT, and functional changes in Cbl-b and c-Cbl might represent the underlying mechanism. AA may be considered for use during LDLT to decrease hyperfibrinolysis.
Assuntos
Ácido Ascórbico/uso terapêutico , Plaquetas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Transplante de Fígado/efeitos adversos , Doadores Vivos/estatística & dados numéricos , Proteína Oncogênica v-cbl/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Trombose/tratamento farmacológico , Antioxidantes/uso terapêutico , Plaquetas/metabolismo , Plaquetas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Oncogênica v-cbl/genética , Fosforilação , Estudos Prospectivos , Proteínas Proto-Oncogênicas c-cbl/genética , Trombose/etiologia , Trombose/metabolismoRESUMO
Aims: The E3-ligase CBL-B (Casitas B-cell lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis. Methods and results: The expression of CBL-B in human atherosclerotic plaques was lower in advanced lesions compared with initial lesions and correlated inversely with necrotic core area. Twenty weeks old Cblb-/-Apoe-/- mice showed a significant increase in plaque area in the aortic arch, where initial plaques were present. In the aortic root, a site containing advanced plaques, lesion area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages due to increased apoptosis, larger necrotic cores, and more CD8+ T cells. Cblb-/-Apoe-/- macrophages exhibited enhanced migration and increased cytokine production and lipid uptake. Casitas B-cell lymphoma-B deficiency increased CD8+ T cell numbers, which were protected against apoptosis and regulatory T cell-mediated suppression. IFNγ and granzyme B production was enhanced in Cblb-/-Apoe-/- CD8+ T cells, which provoked macrophage killing. Depletion of CD8+ T cells in Cblb-/-Apoe-/- bone marrow chimeras rescued the phenotype, indicating that CBL-B controls atherosclerosis mainly through its function in CD8+ T cells. Conclusion: Casitas B-cell lymphoma-B expression in human plaques decreases during the progression of atherosclerosis. As an important regulator of immune responses in experimental atherosclerosis, CBL-B hampers macrophage recruitment and activation during initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques.
Assuntos
Aterosclerose/etiologia , Linfócitos T CD8-Positivos/imunologia , Linfoma de Células B/complicações , Macrófagos/patologia , Proteína Oncogênica v-cbl/metabolismo , Placa Aterosclerótica/etiologia , Animais , Apoptose , Aterosclerose/metabolismo , Aterosclerose/patologia , Modelos Animais de Doenças , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologiaRESUMO
We describe truncation and SAR studies to identify a pentapeptide that binds Cbl tyrosine kinase binding domain with a higher affinity than the parental peptide. The pentapeptide has an alternative binding mode that allows occupancy of a previously uncharacterized groove. A peptide library was used to map the binding site and define the interface landscape. Our results suggest that the pentapeptide is an ideal starting point for the development of inhibitors against Cbl driven diseases.
Assuntos
Modelos Moleculares , Oligopeptídeos/química , Proteína Oncogênica v-cbl/química , Proteínas Tirosina Quinases/química , Sítios de Ligação , Proteína Oncogênica v-cbl/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs.
Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Mutação de Sentido Incorreto , Proteína Oncogênica v-cbl/metabolismo , Osteogênese , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Substituição de Aminoácidos , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Células da Medula Óssea/citologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Oncogênica v-cbl/antagonistas & inibidores , Proteína Oncogênica v-cbl/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células Estromais/citologia , Células Estromais/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genéticaRESUMO
The Cbl protein is a key player in macrophage colony-stimulating factor (M-CSF)-induced signaling. To examine the role of Cbl in M-CSF-mediated cellular events, we used Cbl(YF/YF) knockin mice in which the regulatory tyrosine 737, which when phosphorylated binds to the p85 subunit of phosphatidylinositol 3 kinase (PI3K), is substituted to phenylalanine. In ex vivo cultures, M-CSF and receptor activator of nuclear factor-kappaB ligand-mediated differentiation of bone marrow precursors from Cbl(YF/YF) mice generated increased number of osteoclasts; however, osteoclast numbers in Cbl(YF/YF) cultures were unchanged with increasing doses of M-CSF. We found that Cbl(YF/YF) osteoclasts have enhanced intrinsic ability to survive, and this response was further augmented upon exposure to M-CSF. Treatment of osteoclasts with M-CSF-induced actin reorganization and lamellipodia formation in wild-type osteoclasts; however, in Cbl(YF/YF) osteoclasts lamellipodia formation was compromised. Collectively, these results indicate that abrogation of the Cbl-PI3K interaction, although not affecting M-CSF-induced proliferation and differentiation of precursors, is required for regulation of survival and actin cytoskeletal reorganization of mature osteoclasts.
Assuntos
Fatores Estimuladores de Colônias/farmacologia , Citoesqueleto/efeitos dos fármacos , Proteína Oncogênica v-cbl/metabolismo , Osteoclastos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Remodelação Óssea/genética , Remodelação Óssea/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Proteína Oncogênica v-cbl/genética , Proteína Oncogênica v-cbl/fisiologia , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoclastos/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Tie2 [where 'Tie' is an acronym from tyrosine kinase with Ig and EGF (epidermal growth factor) homology domains] is a receptor tyrosine kinase expressed predominantly on the surface of endothelial cells. Activated by its ligands, the angiopoietins, Tie2 initiates signalling pathways that modulate vascular stability and angiogenesis. Deletion of either the Tie2 or Ang1 (angiopoietin-1) gene in mice results in lethal vascular defects, signifying their importance in vascular development. The mechanism employed by the Tie2 signalling machinery to attenuate or cause receptor trafficking is not well defined. Stimulation of Tie2-expressing cells with Ang1 results in its ubiquitylation, suggesting that this may provide the necessary signal for receptor turnover. Using a candidate molecule approach, we demonstrate that Tie2 co-immunoprecipitates with c-Cbl in an Ang1-dependent manner and its ubiquitylation can be inhibited by the dominant-interfering molecule v-Cbl (a viral form of c-Cbl that contains only the tyrosine kinase-binding domain region). Inhibition of the Tie2-Cbl interaction by overexpression of v-Cbl blocks ligand-induced Tie2 internalization and degradation. In summary, our results illustrate that c-Cbl interacts with the Tie2 signalling complex in a stimulation-dependent manner, and that this interaction is required for Tie2 ubiquitylation, internalization and degradation.
Assuntos
Angiopoietina-1/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptor TIE-2/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologia , Angiopoietina-1/genética , Angiopoietina-1/farmacologia , Linhagem Celular , Humanos , Proteína Oncogênica v-cbl/genética , Proteína Oncogênica v-cbl/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-cbl/genética , Receptor TIE-2/genética , Transdução de Sinais/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
The structure of intrinsic factor (IF) in complex with cobalamin (Cbl) was determined at 2.6-A resolution. The overall fold of the molecule is that of an alpha(6)/alpha(6) barrel. It is a two-domain protein, and the Cbl is bound at the interface of the domains in a base-on conformation. Surprisingly, two full-length molecules, each comprising an alpha- and a beta-domain and one Cbl, and two truncated molecules with only an alpha- domain are present in the same asymmetric unit. The environment around Cbl is dominated by uncharged residues, and the sixth coordinate position of Co(2+) is empty. A detailed comparison between the IF-B12 complex and another Cbl transport protein complex, trans-Cbl-B12, has been made. The pH effect on the binding of Cbl analogues in transport proteins is analyzed. A possible basis for the lack of interchangeability of human and rat IF receptors is presented.
Assuntos
Fator Intrínseco/química , Fator Intrínseco/metabolismo , Vitamina B 12/química , Vitamina B 12/metabolismo , Cristalografia por Raios X , Humanos , Fator Intrínseco/genética , Modelos Moleculares , Proteína Oncogênica v-cbl/química , Proteína Oncogênica v-cbl/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Eletricidade Estática , Homologia Estrutural de ProteínaRESUMO
A high-fat diet (HFD) is associated with reduced glucose uptake in muscle, but not in adipose tissue. In the present study, we investigated whether a HFD can modulate glucose uptake in adipose tissue by increasing signal transduction through the CAP/Cbl pathway, independently of the PI3-K/Akt pathway. Our results suggest that, in HFD, the differential regulation of insulin-induced glucose uptake between skeletal muscle and adipose tissue may, in part, be a consequence of the CAP/Cbl/C3G pathway, since the expression of CAP and Cbl, and also the activation of this pathway were increased in adipose tissue but not in muscle.
Assuntos
Tecido Adiposo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Gorduras na Dieta/administração & dosagem , Proteína Oncogênica v-cbl/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteínas do Citoesqueleto/genética , Glucose/metabolismo , Insulina/metabolismo , Masculino , Músculo Esquelético/metabolismo , Proteína Oncogênica v-cbl/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos WistarRESUMO
Phosphorylation by the constitutively activated BCR-ABL tyrosine kinase is associated with the pathogenesis of the human chronic myelogenous leukemia (CML). It is difficult to characterize kinase response to stimuli or drug treatment because regulatory phosphorylation events are largely transient changes affecting low abundance proteins. Stable isotope labeling with amino acids in cell culture (SILAC) has emerged as a pivotal technology for quantitative proteomics. By metabolically labeling proteins with light or heavy tyrosine, we are able to quantify the change in phosphorylation of BCR-ABL kinase and its substrates in response to drug treatment in human CML cells. In this study, we observed that BCR-ABL kinase is phosphorylated at tyrosines 393 and 644, and that SH2-domain containing inositol phosphatase (SHIP)-2 and downstream of kinase (Dok)-2 are phosphorylated at tyrosine 1135 and 299, respectively. Based on the relative intensity of isotopic peptide pairs, we demonstrate that the level of phosphorylation of BCR-ABL kinase as well as SHIP-2 and Dok-2 is reduced approximately 90% upon treatment with Imatinib, a specific inhibitor of BCR-ABL kinase. Furthermore, proteins, such as SHIP-1, SH2-containing protein (SHC) and Casitas B-lineage lymphoma proto-oncogene (CBL), are also regulated by Imatinib. These results demonstrate the simplicity and utility of SILAC as a method to quantify dynamic changes in phosphorylation at specific sites in response to stimuli or drug treatment in cell culture.
Assuntos
Antineoplásicos/farmacologia , Piperazinas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Benzamidas , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl , Humanos , Mesilato de Imatinib , Marcação por Isótopo , Leucemia Mielogênica Crônica BCR-ABL Positiva , Dados de Sequência Molecular , Proteína Oncogênica v-cbl/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Proto-Oncogene Mas , Tirosina/metabolismoRESUMO
Activating mutations in the epidermal growth factor receptor (EGFR), localized in the activation loop within the kinase domain, have been discovered in non-small cell lung cancers (NSCLC). Most of these mutants are exquisitely sensitive to EGFR tyrosine kinase inhibitors, suggesting that they generate receptor dependence in the cancers that express them. 32D cells stably expressing EGFR-L861Q and EGFR-L858R but not wild-type EGFR exhibited ligand-independent receptor phosphorylation and viability. Ligand-induced receptor down-regulation (LIRD) was impaired in mutant-expressing cells. The EGFR mutants were constitutively associated with the E3 ubiquitin ligase Cbl but did not associate with the adaptor protein CIN85 on the addition of ligand. Inhibition of HSP90 activity with geldanamycin restored Cbl function as indicated by receptor ubiquitination and LIRD. These results suggest that EGFR mutants form defective endocytic complexes. In addition, HSP90 plays a role in maintaining the functional conformation of EGFR mutants and protecting activated receptors from LIRD.
Assuntos
Receptores ErbB/genética , Proteínas de Choque Térmico HSP90/metabolismo , Proteína Oncogênica v-cbl/antagonistas & inibidores , Animais , Sobrevivência Celular/genética , Regulação para Baixo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Ligantes , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Proteína Oncogênica v-cbl/genética , Proteína Oncogênica v-cbl/metabolismo , Transfecção , Transplante Heterólogo , Ubiquitina/metabolismoRESUMO
Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction.
Assuntos
Membrana Celular/fisiologia , Endotelina-1/farmacologia , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Células 3T3-L1 , Animais , Camundongos , Proteínas de Transporte de Monossacarídeos , Proteína Oncogênica v-cbl/metabolismo , Pressão Osmótica , Fosfatidilinositol 4,5-Difosfato/farmacologia , Fosfatidilinositóis/farmacologia , Fosforilação , Transdução de Sinais , Tirosina/metabolismoRESUMO
The kinase inhibitor imatinib is used in the treatment of chronic myeloid leukaemia, where it targets the intracellular Bcr-Abl tyrosine kinase, and gastrointestinal stromal tumours, where it targets either the KIT or PDGF tyrosine kinase receptors. Here, we report that imatinib is also an effective inhibitor of the closely related FMS receptor for macrophage colony stimulating factor and that mutation of Asp 802 of FMS to Val confers imatinib resistance. Imatinib readily reverted the transformed phenotype of haemopoietic and fibroblast cell lines that express the oncogene v-fms and also inhibited the growth of the Bacl.2F5 macrophage cell line. The cellular IC50 value of imatinib for FMS was similar to those for Bcr-Abl and KIT. Consequently, imatinib may also prove effective for the treatment of diseases whose progression is dependent upon macrophage-colony stimulating factor, this includes certain aspects of cancer and inflammation.
Assuntos
Antineoplásicos/farmacologia , Ácido Aspártico/química , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Mutação , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/farmacologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Valina/química , Animais , Benzamidas , Progressão da Doença , Relação Dose-Resposta a Droga , Regulação para Baixo , Humanos , Mesilato de Imatinib , Inflamação , Concentração Inibidora 50 , Camundongos , Neoplasias/metabolismo , Proteína Oncogênica v-cbl/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-crk/metabolismo , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
AIM: To construct the chimeric cblN/Zap molecule, and to observe its effect on TCRzeta protein in pcDNA3.1(+)-cblN/Zab transfected Jurkat cells. METHODS: Total RNA of Jurkat cells were isolated and reversely transcribed into cDNA, which were used as templates to amplify Zap SH2 by PCR. cblN gene tagged with 24 bp flag was amplified by PCR using pEFHAcbl plasmid encoding human cbl as templates. BamH I and EcoR V restriction enzyme digestion sites were introduced into flank SH2 in pcDNA3.1(+) cblN by overlapping extension PCR. Then pcDNA3.1(+)-cblN/Zap was obtained by replacing SH2 of cblN with ZapSH2. After confirmation by enzyme digestion and sequencing, the recombined vector was stably transfected into Jurkat cells with lipofectin. The expression of flag-cblN/Zap was detected by RT-PCR and Western blot, and its effect on TCRzeta protein was analyzed by Western blot. RESULTS: Restriction enzyme digestion analysis of the pcDNA3.1(+)-cblN/Zap recombinant vector showed that the expected fragments were produced and confirmed by sequencing. pcDNA3.1(+)-cblN/Zap was stably transfected into Jurkat cells and the expression of flag-cblN/Zap in the stable clones was confirmed by both RT-PCR and Western blot. The expressed cblN/Zap down-regulated TCRzeta protein in Jurkat cells. CONCLUSION: Chimeric cblN/Zap is able to down-regulate TCRzeta in Jurkat cells.