RESUMO
Acquired uniparental disomy (aUPD) is a common feature of cancer genomes, leading to loss of heterozygosity. aUPD is associated not only with loss-of-function mutations of tumour suppressor genes, but also with gain-of-function mutations of proto-oncogenes. Here we show unique gain-of-function mutations of the C-CBL (also known as CBL) tumour suppressor that are tightly associated with aUPD of the 11q arm in myeloid neoplasms showing myeloproliferative features. The C-CBL proto-oncogene, a cellular homologue of v-Cbl, encodes an E3 ubiquitin ligase and negatively regulates signal transduction of tyrosine kinases. Homozygous C-CBL mutations were found in most 11q-aUPD-positive myeloid malignancies. Although the C-CBL mutations were oncogenic in NIH3T3 cells, c-Cbl was shown to functionally and genetically act as a tumour suppressor. C-CBL mutants did not have E3 ubiquitin ligase activity, but inhibited that of wild-type C-CBL and CBL-B (also known as CBLB), leading to prolonged activation of tyrosine kinases after cytokine stimulation. c-Cbl(-/-) haematopoietic stem/progenitor cells (HSPCs) showed enhanced sensitivity to a variety of cytokines compared to c-Cbl(+/+) HSPCs, and transduction of C-CBL mutants into c-Cbl(-/-) HSPCs further augmented their sensitivities to a broader spectrum of cytokines, including stem-cell factor (SCF, also known as KITLG), thrombopoietin (TPO, also known as THPO), IL3 and FLT3 ligand (FLT3LG), indicating the presence of a gain-of-function that could not be attributed to a simple loss-of-function. The gain-of-function effects of C-CBL mutants on cytokine sensitivity of HSPCs largely disappeared in a c-Cbl(+/+) background or by co-transduction of wild-type C-CBL, which suggests the pathogenic importance of loss of wild-type C-CBL alleles found in most cases of C-CBL-mutated myeloid neoplasms. Our findings provide a new insight into a role of gain-of-function mutations of a tumour suppressor associated with aUPD in the pathogenesis of some myeloid cancer subsets.
Assuntos
Genes Supressores de Tumor , Leucemia Mieloide/genética , Proteínas Mutantes/metabolismo , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Desequilíbrio Alélico , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromossomos Humanos Par 11/genética , Feminino , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Nus , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , Células NIH 3T3 , Transplante de Neoplasias , Oncogenes/genética , Fosforilação , Conformação Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-cbl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-cbl/química , Proteínas Proto-Oncogênicas c-cbl/deficiência , Ubiquitinação , Dissomia Uniparental/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Osteoporosis is a major health problem; however, the mechanisms regulating adult bone mass are poorly understood. Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein that localizes at cell adhesion plaques that form where osteoblasts attach to substrate. To investigate the potential role of CIZ in regulating adult bone mass, we examined the bones in CIZ-deficient mice. Bone volume was increased and the rates of bone formation were increased in CIZ-deficient mice, whereas bone resorption was not altered. CIZ deficiency enhanced the levels of mRNA expression of genes encoding proteins related to osteoblastic phenotypes, such as alkaline phosphatase (ALP) as well as osterix mRNA expression in whole long bones. Bone marrow cells obtained from the femora of CIZ-deficient mice revealed higher ALP activity in culture and formed more mineralized nodules than wild-type cells. CIZ deficiency enhanced bone morphogenetic protein (BMP)-induced osteoblastic differentiation in bone marrow cells in cultures, indicating that BMP is the target of CIZ action. CIZ deficiency increased newly formed bone mass after femoral bone marrow ablation in vivo. Finally, BMP-2-induced bone formation on adult mouse calvariae in vivo was enhanced by CIZ deficiency. These results establish that CIZ suppresses the levels of adult bone mass through inhibition of BMP-induced activation of osteoblasts.
Assuntos
Proteínas Morfogenéticas Ósseas/biossíntese , Calcificação Fisiológica/fisiologia , Regulação para Baixo/fisiologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Transativadores/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Calcificação Fisiológica/genética , Células Cultivadas , Regulação para Baixo/genética , Feminino , Fêmur/fisiologia , Camundongos , Camundongos Knockout , Osteogênese/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator de Transcrição Sp7 , Transativadores/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
Ephrins and Eph receptors are involved in axon guidance and cellular morphogenesis. An interaction between ephrin and Eph receptors elicits neuronal growth-cone collapse through cytoskeletal disassembly. When NIH3T3 cells were plated onto an ephrinA1-coated surface, the cells both adhered and spread. Adhesion and spreading proceeded concomitantly with changes in both the actin and microtubule cytoskeleton. EphA2, focal adhesion kinase (FAK) and p130(cas) were identified as the major ephrin-dependent phosphotyrosyl proteins during the ephrin-induced morphological changes. Mouse embryonic fibroblasts (MEFs) derived from FAK(-/-) and p130(cas-/-) mice had severe defects in ephrinA1-induced cell spreading, which were reversed after re-expression of FAK or p130(cas), respectively. Expression of a constitutively active EphA2 induced NIH3T3 cells to undergo identical, but ligand-independent, morphological changes. These data show that ephrinA1 can induce cell adhesion and actin cytoskeletal changes in fibroblasts in a FAK- and p130(cas)-dependent manner, through activation of the EphA2 receptor. The finding that ephrin Eph signalling can result in actin cytoskeletal assembly, rather than disassembly, has many implications for ephrin Eph responses in other cell types.
Assuntos
Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/farmacologia , Células 3T3 , Actinas/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proteína Substrato Associada a Crk , Proteínas do Citoesqueleto/metabolismo , Efrina-A1 , Efrina-A2 , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Camundongos , Camundongos Knockout , Paxilina , Fosfoproteínas/genética , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor EphA2 , Proteína p130 Retinoblastoma-Like , Transdução de Sinais , Fatores de Transcrição/metabolismo , Tirosina/metabolismoRESUMO
Embryonic development of multilineage hematopoiesis requires the precisely regulated expression of lineage-specific transcription factors, including AML-1 (encoded by Runx1; also known as CBFA-2 or PEBP-2alphaB). In vitro studies and findings in human diseases, including leukemias, myelodysplastic syndromes and familial platelet disorder with predisposition to acute myeloid leukemia (AML), suggest that AML-1 has a pivotal role in adult hematopoiesis. However, this role has not been fully uncovered in vivo because of the embryonic lethality of Runx1 knockout in mice. Here we assess the requirement of AML-1/Runx1 in adult hematopoiesis using an inducible gene-targeting method. In the absence of AML-1, hematopoietic progenitors were fully maintained with normal myeloid cell development. However, AML-1-deficient bone marrow showed inhibition of megakaryocytic maturation, increased hematopoietic progenitor cells and defective T- and B-lymphocyte development. AML-1 is thus required for maturation of megakaryocytes and differentiation of T and B cells, but not for maintenance of hematopoietic stem cells (HSCs) in adult hematopoiesis.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Linfócitos/fisiologia , Megacariócitos/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem da Célula , Tamanho Celular , Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/genética , Marcação de Genes/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Linfócitos/citologia , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genéticaRESUMO
Excessive accumulation of smooth-muscle cells (SMCs) has a key role in the pathogenesis of vascular diseases. It has been assumed that SMCs derived from the outer medial layer migrate, proliferate and synthesize extracellular matrix components on the luminal side of the vessel. Although much effort has been devoted to targeting migration and proliferation of medial SMCs, there is no effective therapy that prevents occlusive vascular remodeling. We show here that in models of post-angioplasty restenosis, graft vasculopathy and hyperlipidemia-induced atherosclerosis, bone-marrow cells give rise to most of the SMCs that contribute to arterial remodeling. Notably, purified hematopoietic stem cells differentiate into SMCs in vitro and in vivo. Our findings indicate that somatic stem cells contribute to pathological remodeling of remote organs, and may provide the basis for the development of new therapeutic strategies for vascular diseases through targeting mobilization, homing, differentiation and proliferation of bone marrow-derived vascular progenitor cells.
Assuntos
Arteriosclerose/etiologia , Arteriosclerose/patologia , Células-Tronco Hematopoéticas/patologia , Músculo Liso Vascular/patologia , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Diferenciação Celular , Divisão Celular , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Oclusão de Enxerto Vascular/etiologia , Oclusão de Enxerto Vascular/patologia , Proteínas de Fluorescência Verde , Técnicas In Vitro , Óperon Lac , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RatosRESUMO
Chronic myelogenous leukemia (CML) is characterized by the presence of the chimeric p210bcr/abl oncoprotein that shows elevated and constitutive protein tyrosine kinase activity relative to the normal c-abl tyrosine kinase. Although several p210bcr/abl substrates have been identified, their relevance in the pathogenesis of the disease is unclear. We have identified a family of proteins, Dok (downstream of tyrosine kinase), coexpressed in hematopoietic progenitor cells. Members of this family such as p62dok (Dok-1) and p56dok-2 (Dok-2) associate with the p120 rasGTPase-activating protein (rasGAP) upon phosphorylation by p210bcr/abl as well as receptor and nonreceptor tyrosine kinases. Here, we report the generation and characterization of single and double Dok-1 or Dok-2 knockout (KO) mutants. Single KO mice displayed normal steady-state hematopoiesis. By contrast, concomitant Dok-1 and Dok-2 inactivation resulted in aberrant hemopoiesis and Ras/MAP kinase activation. Strikingly, all Dok-1/Dok-2 double KO mutants spontaneously developed transplantable CML-like myeloproliferative disease due to increased cellular proliferation and reduced apoptosis. Furthermore, Dok-1 or Dok-2 inactivation markedly accelerated leukemia and blastic crisis onset in Tec-p210bcr/abl transgenic mice known to develop, after long latency, a myeloproliferative disorder resembling human CML. These findings unravel the critical and unexpected role of Dok-1 and Dok-2 in tumor suppression and control of the hematopoietic compartment homeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Crise Blástica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/genética , Crise Blástica/genética , Crise Blástica/patologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica/genética , Hematopoese/genética , Homeostase/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosfoproteínas/genética , Fosforilação , Proteínas de Ligação a RNA/genética , Proteína p120 Ativadora de GTPase/metabolismoRESUMO
Dok-1 and Dok-2 are closely related rasGAP-associated docking proteins expressed preferentially in hematopoietic cells. Although they are phosphorylated upon activation of many protein tyrosine kinases (PTKs), including those coupled with cytokine receptors and oncogenic PTKs like Bcr-Abl, their physiological roles are largely unidentified. Here, we generated mice lacking Dok-1 and/or Dok-2, which included the double-deficient mice succumbed to myeloproliferative disease resembling human chronic myelogenous leukemia (CML) and chronic myelomonocytic leukemia. The double-deficient mice displayed medullary and extramedullary hyperplasia of granulocyte/macrophage progenitors with leukemic potential, and their myeloid cells showed hyperproliferation and hypo-apoptosis upon treatment and deprivation of cytokines, respectively. Consistently, the mutant myeloid cells showed enhanced Erk and Akt activation upon cytokine stimulation. Moreover, loss of Dok-1 and/or Dok-2 induced blastic transformation of chronic phase CML-like disease in mice carrying the bcr-abl gene, a cause of CML. These findings demonstrate that Dok-1 and Dok-2 are key negative regulators of cytokine responses and are essential for myeloid homeostasis and suppression of leukemia.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica/genética , Células Precursoras de Granulócitos/metabolismo , Células Precursoras de Granulócitos/patologia , Homeostase/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mielopoese/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas de Ligação a RNA/genéticaRESUMO
The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.
Assuntos
Toxinas Botulínicas , Proteínas de Transporte/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas , Proteínas Proto-Oncogênicas , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Células 3T3 , ADP Ribose Transferases/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Amidas/farmacologia , Animais , Proteínas de Transporte/genética , Proteína Substrato Associada a Crk , Inibidores Enzimáticos/farmacologia , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Forminas , Expressão Gênica/fisiologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisofosfolipídeos/farmacologia , Camundongos , Mutação/fisiologia , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas c-crk , Piridinas/farmacologia , Proteína p130 Retinoblastoma-Like , Transdução de Sinais/fisiologia , Tirosina/metabolismo , Quinases Associadas a rhoRESUMO
TEL is an ETS family transcription factor that possesses multiple putative mitogen-activated protein kinase phosphorylation sites. We here describe the functional regulation of TEL via ERK pathways. Overexpressed TEL becomes phosphorylated in vivo by activated ERK. TEL is also directly phosphorylated in vitro by ERK. The inducible phosphorylation sites are Ser(213) and Ser(257). TEL binds to a common docking domain in ERK. In vivo ERK-dependent phosphorylation reduces trans-repressional and DNA-binding abilities of TEL for ETS-binding sites. A mutant carrying substituted glutamates on both Ser(213) and Ser(257) functionally mimics hyperphosphorylated TEL and also shows a dominant-negative effect on TEL-induced transcriptional suppression. Losing DNA-binding affinity through phosphorylation but heterodimerizing with unmodified TEL could be an underlying mechanism. Moreover, the glutamate mutant dominantly interferes with TEL-induced erythroid differentiation in MEL cells and growth suppression in NIH 3T3 cells. Finally, endogenous TEL is dephosphorylated in parallel with ERK inactivation in differentiating MEL cells and is phosphorylated through ERK activation in Ras-transformed NIH 3T3 cells. These data indicate that TEL is a constituent downstream of ERK in signal transduction systems and is physiologically regulated by ERK in molecular and biological features.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/genética , Células Eritroides/citologia , Células Eritroides/metabolismo , Humanos , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-ets , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Serina/metabolismo , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
The AML1 (RUNX1) gene, one of the most frequent targets of translocations associated with human leukemias, encodes a DNA-binding protein that plays pivotal roles in myeloid differentiation through transcriptional regulation of various genes. Previously, we reported that AML1 is phosphorylated on two serine residues with dependence on activation of extracellular signal-regulated kinase, which positively regulates the transcriptional activity of AML1. Here, we demonstrate that the interaction between AML1 and the corepressor mSin3A is regulated by phosphorylation of AML1 and that release of AML1 from mSin3A induced by phosphorylation activates its transcriptional activity. Furthermore, phosphorylation of AML1 regulates its intranuclear location and disrupts colocalization of AML1 with mSin3A in the nuclear matrix. PEBP2 beta/CBF beta, a heterodimeric partner of AML1, was shown to play a role in protecting AML1 from proteasome-mediated degradation. We show that mSin3A also protects AML1 from proteasome-mediated degradation and that phosphorylation-induced release of AML1 from mSin3A results in degradation of AML1 in a time-dependent manner. This study provides a novel regulatory mechanism for the function of transcription factors mediated by protein modification and interaction with cofactors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células COS , Núcleo Celular/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/genética , Fator de Crescimento Epidérmico/metabolismo , Fibroblastos/metabolismo , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Complexo Correpressor Histona Desacetilase e Sin3 , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Bcr-Abl kinase is known to reverse apoptosis of cytokine-dependent cells due to cytokine deprivation, although it has been controversial whether chronic myeloid leukemia (CML) progenitors have the potential to survive under conditions in which there are limited amounts of cytokines. Here we demonstrate that early hematopoietic progenitors (Sca-1(+) c-Kit(+) Lin(-)) isolated from normal mice rapidly undergo apoptosis in the absence of cytokines. In these cells, the expression of Bim, a proapoptotic relative of Bcl-2 which plays a key role in the cytokine-mediated survival system, is induced. In contrast, those cells isolated from our previously established CML model mice resist apoptosis in cytokine-free medium without the induction of Bim expression, and these effects are reversed by the Abl-specific kinase inhibitor imatinib mesylate. In addition, the expression levels of Bim are uniformly low in cell lines established from patients in the blast crisis phase of CML, and imatinib induced Bim in these cells. Moreover, small interfering RNA that reduces the expression level of Bim effectively rescues CML cells from apoptosis caused by imatinib. These findings suggest that Bim plays an important role in the apoptosis of early hematopoietic progenitors and that Bcr-Abl supports cell survival in part through downregulation of this cell death activator.
Assuntos
Apoptose/fisiologia , Proteínas de Fusão bcr-abl/metabolismo , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Benzamidas , Biomarcadores , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Células Cultivadas , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Fusão bcr-abl/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Pirimidinas/farmacologia , RNA Interferente Pequeno/metabolismoRESUMO
Evi-1 is a transcription factor that is implicated in leukemic transformation of hematopoietic cells. Two distinct alternative forms, Evi-1a and Evi-1c, are generated from the EVI-1 gene. Whereas Evi-1a is widely recognized as an oncoprotein, a role for Evi-1c, which has an additional PR domain in the amino-terminus of Evi-1a, in leukemogenesis, has not been elucidated thus far. Aberrant oligomerization of transcription factors has recently emerged as a prevalent mechanism for activating their oncogenic potential in hematopoietic malignancies. Here, to study the mechanisms that underlie Evi-1-mediated oncogenesis, we investigated formation of oligomeric complexes by the Evi-1 proteins. We show that Evi-1a forms homo-oligomers, whereas Evi-1c exclusively exists as a monomer in mammalian cells. Remarkably, Evi-1c has lost the ability to interact with CtBP, a transcriptional corepressor that associates with Evi-1a. As a consequence, the ability of Evi-1c to repress transforming growth factor-beta (TGF-beta) signaling is significantly abrogated. These results identify a novel function of a PR domain to regulate oligomerization of transcription factors and suggest that homo-oligomerization may play a critical role in corepressor recruitment by the Evi-1 proteins. In addition, we found that the chimeric oncoprotein acute myelocytic leukemia (AML)1-Evi-1, generated in t(3;21) leukemia, also forms homo-oligomers and hetero-oligomers with Evi-1a, while it did not interact with Evi-1c. Consistent with the results, repression of TGF-beta by AML1-Evi-1 was significantly enhanced by Evi-1a, whereas it was hardly affected by the presence of Evi-1c. These results suggest that oligomerization may contribute to the oncogenic potential of Evi-1-containing proteins.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Fosfoproteínas/metabolismo , Proto-Oncogenes/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Oxirredutases do Álcool , Motivos de Aminoácidos , Animais , Linhagem Celular , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Fosfoproteínas/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/fisiologia , Células Tumorais CultivadasRESUMO
Activating mutations of Fms-like tyrosine kinase 3 (Flt3) are the most common genetic abnormalities found in acute myeloid leukemia (AML) and represent potential therapeutic targets. The novel Flt3 inhibitor KRN383 inhibited the autophosphorylation of Flt3 bearing internal tandem duplications (ITDs) and the Asp835Tyr (D835Y) point mutation with half-maximal inhibitory concentration (IC(50)) values of < or =5.9 and 43 nM, respectively. KRN383 also inhibited the proliferation of the ITD-positive cell lines with IC(50) values of < or =2.9 nM. A single oral administration of 80 mg/kg of KRN383 eradicated ITD-positive xenograft tumors in nude mice and prolonged the survival of SCID mice carrying ITD-positive AML cells. The effectiveness of a single oral dose of KRN383 suggests that it has the potential to be used in a wide variety of clinical regimens, including multicycle and combination therapies.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Mutação Puntual , Inibidores de Proteínas Quinases/administração & dosagem , Quinolinas/farmacologia , Ureia/análogos & derivados , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Administração Oral , Animais , Antimetabólitos Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Estrutura Molecular , Fosforilação , Inibidores de Proteínas Quinases/química , Taxa de Sobrevida , Fatores de Tempo , Ureia/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Invariant natural killer T (iNKT) cells are thought to regulate anti-tumor immunity. Human iNKT (i.e. Valpha24+ NKT) cells have been reported to recognize CD1d on target cells and show cytotoxicity directly on the target cells in vitro. However, the anti-tumor effect of mouse iNKT (i.e. Valpha14+ NKT) cells has been repeatedly reported to be dependent on the activity of natural killer (NK) cells via interferon-gamma, with no evidence of direct cytotoxicity. In the present study, we report that in vitro cytolysis of EL-4 mouse lymphoblastic lymphoma cells by Valpha24+ NKT cells and in vivo eradication of these cells are both dependent on the level of CD1d expression on the tumor cell surface. These observations possibly suggest that direct cytotoxicity of tumor cells by iNKT cells is common to both humans and mice, and that the high expression level of CD1d may be a predictor whether the tumor is a good target of iNKT cells.
Assuntos
Antígenos CD1/biossíntese , Células Matadoras Naturais/metabolismo , Linfócitos T/metabolismo , Animais , Antígenos CD1d , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Galactosilceramidas/metabolismo , Humanos , Imunoterapia/métodos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fenótipo , TransfecçãoRESUMO
CD4+CD25+ regulatory T cells play an important role in peripheral tolerance. These cells have been reported to be capable of suppressing the response of CD4+CD25- T cells in vitro. The depletion of these cells evokes effective immune responses to tumor cells in vivo. In this study, we demonstrate that CD4+CD25+ T cells also suppress all subsets of Valpha24+NKT cells (Valpha24+CD4-CD8- double negative, Valpha24+CD4+, and Valpha24+CD8+) in both proliferation and cytokine production [IFN-gamma, interleukin-4 (IL-4), IL-13, and IL-10]. This suppression is mediated by cell-to-cell contact but not by a humoral factor or the inhibition of antigen-presenting cells. Moreover, the cytotoxic activity of Valpha24+NKT cells against some tumor cell lines is suppressed by CD4+CD25+ T cells. This finding is important in developing an effective immunotherapy for cancer.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Matadoras Naturais/imunologia , Receptores de Interleucina-2/imunologia , Comunicação Celular/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Galactosilceramidas/farmacologia , Humanos , Interferon gama/biossíntese , Interferon gama/metabolismo , Interleucinas/biossíntese , Interleucinas/metabolismo , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Receptores de Interleucina-2/sangue , Subpopulações de Linfócitos T/imunologiaRESUMO
Tumor cells often appear in a deviant differentiated stage, and dedifferentiation is a hallmark of malignancy; however, the causative mechanism of the global changes in dedifferentiation is not understood. The GATA transcription factors function in cell lineage specification during embryonic development and organ formation. The transcriptional targets of the GATA factors in early embryonic development include Disabled-2 and collagen IV, markers for epithelial lineages. GATA-4 and GATA-6 are expressed strongly and are localized in the nucleus in ovarian surface epithelial cells in tissues or primary cell cultures. By immunohistochemistry, we found that 82% of the 50 tumors analyzed had lost GATA-6 function, either by a complete absence of expression or by cytoplasmic mislocalization. The frequent loss of GATA-6 was also confirmed in a panel of ovarian surface epithelial and tumor cell lines. Although GATA-4 is absent only in a small percentage (14%) of ovarian tumors, it is lost in the majority of established cell lines in culture. The loss of GATA-6 correlates with the loss of Disabled-2, collagen IV, and laminin, markers for epithelial cell types. Loss of GATA factors was also found in an in vitro model for spontaneous transformation of rat ovarian epithelial cells. Repression of GATA-6 by small interfering (si)RNA approach in cultured cells leads to dedifferentiation as indicated by the loss of Disabled-2 and laminin expression. Restoration of GATA factors expression by ectopic transfection suppresses cell growth and is incompatible with the maintenance of the cells in culture. However, restoration of GATA-4 and GATA-6 expression is not able to induce expression of endogenous Disabled-2 in tumor cells, suggesting that the loss of GATA factors and dedifferentiation are irreversible processes. In conclusion, we observed the inappropriate expression and cellular localization of the GATA transcription factors in ovarian tumor tissues and cancer cell lines, and we have demonstrated that down-regulation of GATA factor expression leads to dedifferentiation. We propose that alterations of GATA transcription factor expression and aberrant nucleocytoplasmic localization may contribute to the anomalous epithelial dedifferentiation of the ovarian tumor cells.
Assuntos
Proteínas de Ligação a DNA/análise , Neoplasias Ovarianas/química , Fatores de Transcrição/análise , Adulto , Idoso , Diferenciação Celular , Linhagem da Célula , Núcleo Celular/química , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo , Células Epiteliais/citologia , Feminino , Fator de Transcrição GATA4 , Fator de Transcrição GATA6 , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Fatores de Transcrição/fisiologiaRESUMO
OBJECTIVE: For the study of the function of genes of interest in hematopoietic stem cells (HSCs) and for successful gene therapy, it is fundamental to develop a method of efficient gene transfer into HSCs. In mice experiments, efforts have been made to raise the transduction efficiency by modifying the vectors, administrating 5-fluorouracil (5-FU) to donor mice, selecting cytokine cocktails to better sustain the long-term repopulating potential of the stem cells, and so on. The objective of this study is to examine whether the use of fibroblast growth factor-1 (FGF-1)-expanded bone marrow cells provide an improved source for retroviral gene delivery to HSCs. MATERIALS AND METHODS: Unfractionated bone marrow cells from one mouse were cultured in serum-free medium containing FGF-1. Both floating and attached cells were transferred to retronectin precoated dishes and infected with virus supernatant from MP34 cells stably transduced with pMY/GFP retrovirus. After 3-day infection, the green fluorescence protein-positive fraction was sorted and the cells were transplanted to lethally irradiated mice. RESULTS: The experiments illustrated that the number of bone marrow-derived competitive repopulation units (CRUs) was increased from 600 to 9300 per mouse after a 3-week culture period with FGF-1. Following retroviral transduction of the expanded cells, the absolute number of sorted retrovirus-transduced CRUs was 4200. Using these retrovirus-transduced cells in noncompetitive reconstitution assay, we achieved radiation protection and long-term bone marrow reconstitution in 100% of the recipients with average myeloid and lymphoid chimerisms of 70% and 50%, respectively, even if we transplanted 150 recipients with cells derived from a single donor mouse. CONCLUSION: In conclusion, FGF-1-expanded bone marrow cells constitute an excellent source of stem cells that could be used in a range of gene delivery protocols.
Assuntos
Fator 1 de Crescimento de Fibroblastos/farmacologia , Células-Tronco Hematopoéticas/citologia , Retroviridae/genética , Transdução Genética/métodos , Animais , Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Linhagem da Célula , Proliferação de Células/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos C57BLRESUMO
AML1/Evi-1 is a chimeric protein that is derived from t(3;21), found in blastic transformation of chronic myelogenous leukemia. It is composed of the N-terminal AML1 portion with the DNA-binding Runt domain and the C-terminal Evi-1 portion. It has been shown to dominantly repress AML1-induced transactivation. The mechanism for it has been mainly attributed to competition with AML1 for the DNA-binding and for the interaction with PEBP2beta (CBFbeta), a partner protein which heterodimerizes with AML1. It was recently found that Evi-1 interacts with C-terminal binding protein (CtBP) to repress TGFbeta-induced transactivation. Here, we demonstrate that AML1/Evi-1 interacts with CtBP in SKH1 cells, a leukemic cell line which endogenously overexpresses AML1/Evi-1 and that AML1/Evi-1 requires the interaction with CtBP to repress AML1-induced transactivation. The association with CtBP is also required when AML1/Evi-1 blocks myeloid differentiation of 32Dcl3 cells induced by granulocyte colony-stimulating factor. Taken together, it is suggested that one of the mechanisms for AML1/Evi-1-associated leukemogenesis should be an aberrant recruitment of a corepressor complex by the chimeric protein.
Assuntos
Fusão Gênica Artificial , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 3/genética , Proteínas de Ligação a DNA/metabolismo , Leucemia Mieloide/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas , Proto-Oncogenes , Fatores de Transcrição/metabolismo , Oxirredutases do Álcool , Animais , Sítios de Ligação , Northern Blotting , Western Blotting , Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core , Cricetinae , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Regulação Neoplásica da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/farmacologia , Granulócitos/citologia , Granulócitos/metabolismo , Histona Desacetilases/metabolismo , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Testes de Precipitina , RNA/metabolismo , Deleção de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , Transcrição GênicaRESUMO
TEL belongs to a member of the ETS family transcription factors that represses transcription of target genes such as FLI-1. Although TEL is essential for establishing hematopoiesis in neonatal bone marrow, its role in erythroid lineage is not understood. To investigate a role for TEL in erythroid differentiation, we introduced TEL into mouse erythroleukemia (MEL) cells. Overexpressing wild-type-TEL in MEL cells enhanced differentiation induced by hexamethylene bisacetamide or dimethylsulfoxide, as judged by the increased levels of erythroid-specific delta-aminolevulinate synthase and beta-globin mRNAs. TEL bound to a corepressor mSin3A through the helix-loop-helix domain. A TEL mutant lacking this domain still bound to the ETS binding site, but lost its transrepressional effect. This mutant completely blocked erythroid differentiation in MEL cells. Moreover, it showed dominant-negative effects over TEL-mediated transcriptional repression and acceleration of erythroid differentiation. Endogenous TEL mRNA was found to increase during the first 3 days in differentiating MEL cells and drastically decrease thereafter. All these data suggest that TEL might play some role in erythroid cell differentiation.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Vírus da Leucemia Murina de Friend/isolamento & purificação , Leucemia Eritroblástica Aguda/patologia , Proteínas Repressoras/fisiologia , 5-Aminolevulinato Sintetase/genética , Animais , Sequência de Bases , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Globinas/genética , Células HeLa , Humanos , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/virologia , Camundongos , Proteínas Proto-Oncogênicas c-ets , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
BACKGROUND: Alemtuzumab, a humanized monoclonal antibody directed against human CD52, has a strong lympholytic effect. This study evaluates the safety of unmanipulated peripheral blood stem-cell transplantation from two or three loci-mismatched related donors using alemtuzumab in vivo. METHODS: A total body irradiation-based regimen was used in young patients, whereas those 50 years or older received fludarabine-based conditioning. Alemtuzumab was added to these regimens by intravenous infusion at 0.2 mg/kg per day for 6 days (days -8 to -3). RESULTS: We treated 12 patients with a median age of 49.5 years. Eight patients demonstrated active disease, and four patients demonstrated acute leukemia in high-risk remission. All achieved neutrophil engraftment a median of 17.5 days after transplantation with complete donor-type chimerism. The cumulative incidence of grades III to IV acute graft-versus-host disease was only 9%. Infection-related deaths were not observed. CD3+/CD4+ and CD3+/CD8+ T cells were strongly suppressed within 2 months after transplantation, but recovered on day 90. Relapse was observed in five of eight patients who underwent transplantation for active disease, whereas none of the three patients who underwent transplantation in first remission had a relapse. CONCLUSIONS: We conclude that in vivo alemtuzumab enables haploidentical hematopoietic stem-cell transplantation without ex vivo graft manipulation.