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1.
Lancet Oncol ; 16(7): 763-74, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26045340

RESUMO

BACKGROUND: Necitumumab is a second-generation, recombinant, human immunoglobulin G1 EGFR antibody. In this study, we aimed to compare treatment with necitumumab plus gemcitabine and cisplatin versus gemcitabine and cisplatin alone in patients with previously untreated stage IV squamous non-small-cell lung cancer. METHODS: We did this open-label, randomised phase 3 study at 184 investigative sites in 26 countries. Patients aged 18 years or older with histologically or cytologically confirmed stage IV squamous non-small-cell lung cancer, with an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 and adequate organ function and who had not received previous chemotherapy for their disease were eligible for inclusion. Enrolled patients were randomly assigned centrally 1:1 to a maximum of six 3-week cycles of gemcitabine and cisplastin chemotherapy with or without necitumumab according to a block randomisation scheme (block size of four) by a telephone-based interactive voice response system or interactive web response system. Chemotherapy was gemcitabine 1250 mg/m(2) administered intravenously over 30 min on days 1 and 8 of a 3-week cycle and cisplatin 75 mg/m(2) administered intravenously over 120 min on day 1 of a 3-week cycle. Necitumumab 800 mg, administered intravenously over a minimum of 50 min on days 1 and 8, was continued after the end of chemotherapy until disease progression or intolerable toxic side-effects occurred. Randomisation was stratified by ECOG performance status and geographical region. Neither physicians nor patients were masked to group assignment because of the expected occurrence of acne-like rash--a class effect of EGFR antibodies--that would have unmasked most patients and investigators to treatment. The primary endpoint was overall survival, analysed by intention to treat. We report the final clinical analysis. This study is registered with ClinicalTrials.gov, number NCT00981058. FINDINGS: Between Jan 7, 2010, and Feb 22, 2012, we enrolled 1093 patients and randomly assigned them to receive necitumumab plus gemcitabine and cisplatin (n=545) or gemcitabine and cisplatin (n=548). Overall survival was significantly longer in the necitumumab plus gemcitabine and cisplatin group than in the gemcitabine and cisplatin alone group (median 11·5 months [95% CI 10·4-12·6]) vs 9·9 months [8·9-11·1]; stratified hazard ratio 0·84 [95% CI 0·74-0·96; p=0·01]). In the necitumumab plus gemcitabine and cisplatin group, the number of patients with at least one grade 3 or worse adverse event was higher (388 [72%] of 538 patients) than in the gemcitabine and cisplatin group (333 [62%] of 541), as was the incidence of serious adverse events (257 [48%] of 538 patients vs 203 [38%] of 541). More patients in the necitumumab plus gemcitabine and cisplatin group had grade 3-4 hypomagnesaemia (47 [9%] of 538 patients in the necitumumab plus gemcitabine and cisplatin group vs six [1%] of 541 in the gemcitabine and cisplatin group) and grade 3 rash (20 [4%] vs one [<1%]). Including events related to disease progression, adverse events with an outcome of death were reported for 66 (12%) of 538 patients in the necitumumab plus gemcitabine and cisplatin group and 57 (11%) of 541 patients in the gemcitabine and cisplatin group; these were deemed to be related to study drugs in 15 (3%) and ten (2%) patients, respectively. Overall, we found that the safety profile of necitumumab plus gemcitabine and cisplatin was acceptable and in line with expectations. INTERPRETATION: Our findings show that the addition of necitumumab to gemcitabine and cisplatin chemotherapy improves overall survival in patients with advanced squamous non-small-cell lung cancer and represents a new first-line treatment option for this disease. FUNDING: Eli Lilly and Company.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Cisplatino/administração & dosagem , Intervalo Livre de Doença , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Gentamicinas/administração & dosagem , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Medição de Risco , Fatores Sexuais , Análise de Sobrevida , Resultado do Tratamento
2.
Methods Enzymol ; 404: 411-22, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16413287

RESUMO

The Arf-GTPase-activating protein (GAP) ASAP1 has been identified in a yeast-two-hybrid screen as a prominent binder of the proline-rich tyrosine kinase 2 (Pyk2) via SH3 domain-mediated interaction. Following binding, Pyk2 directly phosphorylates ASAP1 on tyrosine residues 308 and 782 in vitro and in cells. To understand the functional impact of this interaction and subsequent phosphorylation, nonphosphorylated and Pyk2-phosphorylated ASAP1 have been generated. This material can be used for lipid-protein overlay assays and fluorimetric Arf-GTPase tests to show that the Pyk2-mediated tyrosine phosphorylation of ASAP1 modulates its GAP activity towards Arf1 in vitro. These studies provide the first evidence for a regulation of ASAP1 and hence Arf1 activity by tyrosine phosphorylation and suggest a functional link between tyrosine kinases and Arf GTPases. Furthermore, an assay to study cellular Arf activation and thus GAP as well as guanine nucleotide exchange factor (GEF) activities and their regulation is described.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Células Cultivadas , GTP Fosfo-Hidrolases/análise , Humanos , Lipossomos/metabolismo , Ácido Mirístico/metabolismo , Fosfolipídeos/metabolismo , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de src
3.
Mol Biol Cell ; 20(11): 2684-96, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19369422

RESUMO

In metaphase Xenopus egg extracts, global microtubule growth is mainly promoted by two unrelated microtubule stabilizers, end-binding protein 1 (EB1) and XMAP215. Here, we explore their role and potential redundancy in the regulation of spindle assembly and function. We find that at physiological expression levels, both proteins are required for proper spindle architecture: Spindles assembled in the absence of EB1 or at decreased XMAP215 levels are short and frequently multipolar. Moreover, the reduced density of microtubules at the equator of DeltaEB1 or DeltaXMAP215 spindles leads to faulty kinetochore-microtubule attachments. These spindles also display diminished poleward flux rates and, upon anaphase induction, they neither segregate chromosomes nor reorganize into interphasic microtubule arrays. However, EB1 and XMAP215 nonredundantly regulate spindle assembly because an excess of XMAP215 can compensate for the absence of EB1, whereas the overexpression of EB1 cannot substitute for reduced XMAP215 levels. Our data indicate that EB1 could positively regulate XMAP215 by promoting its binding to the microtubules. Finally, we show that disruption of the mitosis-specific XMAP215-EB1 interaction produces a phenotype similar to that of either EB1 or XMAP215 depletion. Therefore, the XMAP215-EB1 interaction is required for proper spindle organization and chromosome segregation in Xenopus egg extracts.


Assuntos
Segregação de Cromossomos , Proteínas Associadas aos Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Extratos Celulares , Feminino , Imunofluorescência , Imunoprecipitação , Microscopia de Fluorescência/métodos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Oócitos/citologia , Ligação Proteica , Xenopus , Proteínas de Xenopus/genética
4.
J Biol Chem ; 284(2): 1324-36, 2009 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-19010788

RESUMO

Prostaglandin D(2) activation of the seven-transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases, such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expression of human CRTH2. We tested the hypothesis that CRTH2 may take advantage of its C-tail to silence its own signaling and that this mechanism may explain the poor functional responses observed with CRTH2 in heterologous expression systems. Although the C terminus is a critical determinant for retention of CRTH2 at the plasma membrane, the presence of this domain confers a signaling-compromised conformation onto the receptor. Indeed, a mutant receptor lacking the major portion of its C-terminal tail displays paradoxically enhanced Galpha(i) and ERK1/2 activation despite enhanced constitutive and agonist-mediated internalization. Enhanced activation of Galpha(i) proteins and downstream signaling cascades is probably due to the inability of the tail-truncated receptor to recruit beta-arrestin2 and undergo homologous desensitization. Unexpectedly, CRTH2 is not phosphorylated upon agonist-stimulation, a primary mechanism by which GPCR activity is regulated. Dynamic mass redistribution assays, which allow label-free monitoring of all major G protein pathways in real time, confirm that the C terminus inhibits Galpha(i) signaling of CRTH2 but does not encode G protein specificity determinants. We propose that intrinsic CRTH2 inhibition by its C terminus may represent a rather unappreciated strategy employed by a GPCR to specify the extent of G protein activation and that this mechanism may compensate for the absence of the classical phosphorylation-dependent signal attenuation.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Arrestinas/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Dados de Sequência Molecular , Fosforilação , Prostaglandina D2/farmacologia , Ligação Proteica , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores de Prostaglandina/química , Receptores de Prostaglandina/genética , Alinhamento de Sequência , Especificidade por Substrato , Fatores de Tempo , Ativação Transcricional/efeitos dos fármacos , beta-Arrestinas
5.
J Biol Chem ; 278(32): 29560-70, 2003 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-12771146

RESUMO

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Tirosina Quinases/fisiologia , Animais , Linhagem Celular , DNA Complementar/metabolismo , Quinase 2 de Adesão Focal , Glutationa Transferase/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Mutagênese Sítio-Dirigida , Células PC12 , Mapeamento de Peptídeos , Fosforilação , Testes de Precipitina , Prolina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Tirosina/química , Domínios de Homologia de src
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