RESUMO
Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors.
Assuntos
Matriz Extracelular/metabolismo , Mutação , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/etiologia , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Animais , Crizotinibe , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/fisiologiaAssuntos
Regulação da Expressão Gênica , Mutação/genética , Síndromes Mielodisplásicas/metabolismo , Transtornos Mieloproliferativos/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Adulto , Sequência de Aminoácidos , Sequência de Bases , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/genética , Prognóstico , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
E2A-hepatic leukemia factor (HLF) is a chimeric protein found in B-lineage acute lymphoblastic leukemia (ALL) with t(17;19). To analyze the leukemogenic process and to create model mice for t(17;19)-positive leukemia, we generated inducible knock-in (iKI) mice for E2A-HLF. Despite the induced expression of E2A-HLF in the hematopoietic tissues, no disease was developed during the long observation period, indicating that additional gene alterations are required to develop leukemia. To elucidate this process, E2A-HLF iKI and control littermates were subjected to retroviral insertional mutagenesis. Virus infection induced acute leukemias in E2A-HLF iKI mice with higher morbidity and mortality than in control mice. Inverse PCR detected three common integration sites specific for E2A-HLF iKI leukemic mice, which induced overexpression of zinc-finger transcription factors: growth factor independent 1 (Gfi1), zinc-finger protein subfamily 1A1 isoform a (Zfp1a1, also known as Ikaros) and zinc-finger protein 521 (Zfp521). Interestingly, tumors with Zfp521 integration exclusively showed B-lineage ALL, which corresponds to the phenotype of human t(17;19)-positive leukemia. In addition, ZNF521 (human counterpart of Zfp521) was found to be overexpressed in human leukemic cell lines harboring t(17;19). Moreover, both iKI for E2A-HLF and transgenic for Zfp521 mice frequently developed B-lineage ALL. These results indicate that a set of transcription factors promote leukemic transformation of E2A-HLF-expressing hematopoietic progenitors and suggest that aberrant expression of Zfp521/ZNF521 may be clinically relevant to t(17;19)-positive B-lineage ALL.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Ligação a DNA/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Transformação Celular Neoplásica , Humanos , Camundongos , Mutação , Proteínas Nucleares , Ativação Transcricional/genéticaRESUMO
Chronic myelogenous leukemia (CML) is a hematopoietic disorder, which begins as indolent chronic phase but inevitably progresses to fatal blast crisis. p210BCR/ABL, a constitutively active tyrosine kinase, is responsible for disease initiation but molecular mechanism(s) underlying disease evolution remains largely unknown. To explore this process, we employed retroviral insertional mutagenesis to CML-exhibiting p210BCR/ABL transgenic mice (Tg). Virus infection induced acute lymphoblastic leukemia (ALL) in p210BCR/ABL Tg with a higher frequency and in a shorter latency than wild-type littermates, and inverse PCR detected two retrovirus common integration sites (CISs) in p210BCR/ABL Tg tumors. Interestingly, one CIS was the transgene itself, where retrovirus integrations induced upregulation of p210BCR/ABL and production of truncated BCR/ABL with an enhanced kinase activity. Another CIS was Notch1 gene, where retrovirus integrations resulted in overexpression of Notch1 and generation of Notch1 lacking the C-terminal region (Notch1DeltaC) associated with stable expression of its activated product, C-terminal-truncated Notch intracellular domain (NICD Delta C). In addition, generation of Tg for both p210BCR/ABL and Notch1DeltaC developed ALL in a shortened period with Stat5 activation, demonstrating the cooperative oncogenicity of Notch1DeltaC/NICD Delta C with p210BCR/ABL involving Stat5-mediated pathway. These results demonstrated that overexpression/enhanced kinase activity of BCR/ABL and altered expression of Notch1 induces acute leukemia in a transgenic model for CML.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiologia , Receptor Notch1/genética , Integração Viral/fisiologia , Animais , Animais Recém-Nascidos , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B/virologia , Northern Blotting , Southern Blotting , Western Blotting , Feminino , Citometria de Fluxo , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vírus do Sarcoma Murino de Moloney/patogenicidade , Mutagênese Insercional/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptor Notch1/metabolismo , Retroviridae/fisiologia , Fator de Transcrição STAT5/metabolismo , Taxa de Sobrevida , Linfócitos T/metabolismo , Linfócitos T/patologia , Linfócitos T/virologiaRESUMO
Initial biochemical signaling originating from high-affinity immunoglobulin E receptor (FcepsilonRI) has been ascribed to Src family kinases. To understand the mechanisms by which individual kinases drive the signaling, we conducted reconstitution experiments: FcepsilonRI signaling in RBL2H3 cells was first suppressed by a membrane-anchored, gain-of-function C-terminal Src kinase and then reconstructed with Src family kinases whose C-terminal negative regulatory sequence was replaced with a c-myc epitope. Those constructs derived from Lyn and Fyn, which are associated with detergent-resistant membranes (DRMs), physically interacted with resting FcepsilonRI and reconstructed clustering-induced signaling that leads to calcium mobilization and ERK1 and -2 activation. c-Src-derived construct, which was excluded from DRMs, failed to interact with FcepsilonRI and to restore the signaling, whereas creation of palmitoylatable Cys3 enabled it to interact with DRMs and with FcepsilonRI and to restore the signaling. Deletion of Src homology 3 (SH3) domain from the Lyn-derived construct did not alter its ability to transduce the series of signaling. Deletion of SH2 domain did not affect its association with DRMs and with FcepsilonRI nor clustering-induced tyrosine phosphorylation of FcepsilonRI beta and gamma subunits, but it almost abrogated the next step of tyrosine phosphorylation of Syk and its recruitment to FcepsilonRI. These findings suggest that Lyn and Fyn could, but c-Src could not, drive FcepsilonRI signaling and that N-terminal palmitoylation and SH2 domain are required in sequence for the initial interaction with FcepsilonRI and for the signal progression to the molecular assembly.
Assuntos
Receptores de IgE/metabolismo , Transdução de Sinais , Domínios de Homologia de src , Quinases da Família src/metabolismo , Animais , Linhagem Celular , Humanos , Ácido Palmítico , Quinases da Família src/genéticaRESUMO
In the repair process after lung injury, the regeneration of alveolar epithelial cells plays an important role by covering the damaged alveolar wall and preventing the activated fibroblasts from invading the intra- alveolar spaces. Hepatocyte growth factor (HGF) is a potent mitogen for alveolar epithelial cells and has been reported to be capable of repressing the fibrosing process by connecting to the c-Met/HGF receptor on alveolar epithelial cells. However, it has been reported that the c-Met expression was downregulated in an acute phase of lung injury, which may limit the effect of HGF for therapeutic use. In the present study we observed that interferon (IFN)-gamma upregulates the c-Met messenger RNA (mRNA) and protein expression in A549 alveolar epithelial cells. We analyzed the mechanism of this upregulation and found that IFN-gamma enhances the transcription of the c-met proto-oncogene, and that it does not prolong the stability of the c-Met mRNA. HGF is known to act as a motogen as well as a mitogen for epithelial cells. We also found that the migratory activity of A549 cells induced by HGF is strongly enhanced by preincubation with IFN-gamma. Finally, we administered recombinant IFN-gamma to C57BL/6 mice and confirmed that this upregulation is also observed in vivo. These results suggest that the combination of HGF and IFN-gamma could be a new therapeutic approach for fibrosing pulmonary diseases.
Assuntos
Interferon gama/farmacologia , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Animais , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Lesão Pulmonar , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proto-Oncogene Mas , Proto-Oncogenes/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Regulação para Cima/efeitos dos fármacosRESUMO
As an attempt to analyze the roles of C-terminal Src kinase (Csk) in the high affinity IgE receptor (FcepsilonRI)-mediated signaling, we overexpressed Csk, a membrane-targeted form of Csk (mCsk), and a kinase-defective, membrane-targeted form of Csk (mCsk(-)) in rat basophil leukemia (RBL) 2H3 cells. Specific activity of Lyn at the basal state was decreased in Csk-expressing cells, and further decreased in mCsk-expressing cells. In mCsk(-)-expressing cells, basal specific activity of Lyn was increased, thereby indicating that mCsk(-) functioned as a dominant negative molecule. The onset of FcepsilonRI-mediated Lyn activation was delayed in Csk-expressing cells, and further delayed in mCsk-expressing cells. In mCsk(-)-expressing cells, Lyn activation was rapid and quite long lasting. These findings indicate (i) Csk negatively regulates rapid FcepsilonRI/Lyn coupling, and (ii) Csk activity is potentially required for its termination. The onsets of the series of events including tyrosyl phosphorylation of Syk, mitogen-activated protein (MAP) kinase activation, elevation of intracellular calcium concentration ([Ca2+]i), and histamine release were all stepwisely delayed in Csk-expressing cells and in mCsk-expressing cells. The durations of Syk phosphorylation and MAP kinase activation also closely correlated with those of Lyn activation, but [Ca2+]i elevation and histamine release followed different temporal patterns: the delayed responses in Csk-expressing cells and in mCsk-expressing cells led to sustained [Ca2+]i oscillation and histamine release, while the prompt responses in parent cells and mCsk(-)-expressing cells rapidly subsided. These findings provide further evidence that the initiations of the FcepsilonRI-mediated signals are upstreamly regulated by Src family protein tyrosine kinases and revealed that their terminations are regulated by Lyn-dependent (Syk and MAP kinase) and -independent ([Ca2+]i elevation and histamine release) mechanisms.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Proteína Tirosina Quinase CSK , Cálcio/metabolismo , Linhagem Celular , Ativação Enzimática , Cinética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Ratos , Quinases da Família src/metabolismoRESUMO
Anti-12(S)-hydroxyeicosatetraenoic acid (12-HETE)-antibody and anti-thromboxane B2 (TXB2)-antibody were generated and applied to the radioimmunoassay. The detection limit for 12-HETE was 16 pg. The cross-reactivities of anti-12-HETE-antibody were 4.6% for 15-HETE, 0.18% for 5-HETE and below 0.15% for leukotrienes and prostaglandins (PGs). 12-HETE and TXB2 released from guinea pig platelets were measured by radioimmunoassay. Platelet activating factor (PAF) at 10(-9) M induced the aggregation of platelets, the releases of immunoreactive-12-HETE (1.8 +/- 1.2 ng/10(8) platelets, mean +/- S.D.) and immunoreactive-TXB2 (18.5 +/- 17.3 ng/10(8) platelets). Collagen at 1 microgram/ml also evoked platelet aggregation, the releases of immunoreactive-12-HETE (2.7 +/- 1.1 ng/10(8) platelets) and immunoreactive-TXB2 (11.8 +/- 4.6 ng/10(8) platelets). By the stimulation with these compounds, TXB2 was produced in a greater amount than 12-HETE from guinea pig platelets. Although 10(-7) M and 10(-6) M U46619, a TXA2 mimetic, caused platelet aggregation, arachidonic acid metabolites were not released. These data suggest the presence of different mechanisms of platelet activation depending on each stimulus.