RESUMEN
Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony-stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated (T) factors (T-granulocyte-macrophage-CSF (T-GM-CSF)) and T-interleukin-3 (T-IL-3)) on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and -IL-3 had enhanced receptor binding, but decreased CSF activity, compared with their FL forms. Importantly, T-GM-CSF and -IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and -IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster-forming cells from patients with acute myeloid leukemia regardless of cytogenetic or molecular alterations and in vivo using animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared with their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-3/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Proliferación Celular , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Unión ProteicaAsunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Leucemia Mieloide Aguda/patología , Proteínas de Fusión Oncogénica/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Proteína 1 Compañera de Translocación de RUNX1/fisiología , Animales , Línea Celular Tumoral , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Ratones , Proteínas Tirosina Fosfatasas/genética , Tasa de SupervivenciaAsunto(s)
Inhibidores Enzimáticos/farmacología , Leucemia Mieloide Aguda/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Factor de Transcripción STAT5/metabolismo , Quinasa Syk/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/fisiología , Secuencia de Aminoácidos , Proliferación Celular , Humanos , Leucemia Mieloide Aguda/metabolismo , Homología de Secuencia de Aminoácido , Tirosina Quinasa 3 Similar a fms/químicaRESUMEN
Internal tandem duplications (ITDs) in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in acute myeloid leukemia (AML). We hypothesized that increased recruitment of the protein tyrosine phosphatase, Shp2, to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and STAT5 activation. Co-immunoprecipitation demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Knockdown of Shp2 in Baf3/N51-FLT3 cells significantly reduced proliferation while having little effect on WT-FLT3-expressing cells. Consistently, mutation of N51-FLT3 tyrosine 599 to phenylalanine or genetic disruption of Shp2 in N51-FLT3-expressing bone marrow low-density mononuclear cells reduced proliferation and STAT5 activation. In transplants, genetic disruption of Shp2 in vivo yielded increased latency to and reduced severity of FLT3-ITD-induced malignancy. Mechanistically, Shp2 co-localizes with nuclear phospho-STAT5, is present at functional interferon-γ activation sites (GAS) within the BCL2L1 promoter, and positively activates the human BCL2L1 promoter, suggesting that Shp2 works with STAT5 to promote pro-leukemogenic gene expression. Further, using a small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was reduced in a dose-dependent manner. These findings demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to AML.
Asunto(s)
Proliferación Celular , Células Madre Hematopoyéticas/citología , Leucemia Mieloide Aguda/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/fisiología , Secuencias Repetidas en Tándem/genética , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Secuencia de Bases , Western Blotting , Trasplante de Médula Ósea , Inmunoprecipitación de Cromatina , Técnica del Anticuerpo Fluorescente , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunoprecipitación , Indoles/farmacología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Mutación/genética , Fosforilación/efectos de los fármacos , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/efectos de los fármacos , Células Precursoras de Linfocitos B/metabolismo , Regiones Promotoras Genéticas/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Tasa de Supervivencia , Triazoles/farmacología , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) pi, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.
Asunto(s)
Resistencia a Antineoplásicos/fisiología , Leucemia Mieloide/enzimología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Enfermedad Aguda , Células de la Médula Ósea/patología , División Celular , Daunorrubicina , Resistencia a Múltiples Medicamentos/fisiología , Células HL-60 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transducción de SeñalRESUMEN
Tyrosine kinase oncogenes such as p210BCR-ABL activate multiple signal pathways. As a result, it is difficult to infer the functional relevance of a pathway acting alone or in cooperation with another. One or 2 second-tier kinases represented in the p21ras and phosphatidylinositol-3-kinase (PI-3-kinase) pathways (activated RafCAAX and gag-akt, respectively) were expressed in parental H7 interleukin-3 (IL-3)-dependent myeloid cells. IL-3-dependent cells served, independently, as recipients of p210BCR-ABL, which activated p21ras and PI-3-kinase pathways, including raf/erk and akt, respectively, en route to transformation. By contrast, neither RafCAAX nor gag-akt when expressed in parental cells in isolation produced factor-independent cells. On the other hand, H7 cells expressing both RafCAAX and gag-akt (H7gag-akt/RafCAAX) were transformed. Such transformation in H7gag-akt/RafCAAX was accomplished in the absence of active versions of Shc or cbl, and there was no evidence of Stat activity and only modest amounts of bcl-xL, a Stat5 transcriptional target protein, all of which characterized the cells transformed by BCR-ABL. However, H7gag-akt/RafCAAX cells and H7BCR-ABL cells cultured in the absence of IL-3 shared strikingly increased p65 nuclear factor kappaB (NFkappaB) activity. Treatment of cells with a specific NFkappaB inhibitor, parthenolide, led to loss of NFkappaB activity and down-regulation of antiapoptotic c-IAP2. In cells with only gag-akt/RafCAAX, this was sufficient to allow polyADP ribosyltransferase (PARP)-degradative apoptosis, but in cells with p210BCR-ABL, apoptosis was blocked, possibly by a Stat5/bcl-xL-dependent mechanism. Therefore, one hematopoietic antiapoptotic program, among others, available to certain tyrosine kinase oncogenes involves a cooperative response between raf/erk and akt, unambiguous components of p21ras and PI-3-kinase pathways, to induce p65 NFkappaB and c-IAP2.
Asunto(s)
Interleucina-3/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis , División Celular , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Vectores Genéticos , Humanos , Proteínas Inhibidoras de la Apoptosis , Leucemia Mieloide , Ratones , FN-kappa B/antagonistas & inhibidores , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes/metabolismo , Virus 40 de los Simios/genética , Transfección , Células Tumorales Cultivadas , Proteínas Virales/metabolismoRESUMEN
The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is associated with transforming ability and an intrinsic resistance to induction of apoptosis by genotoxic agents. Arachidonic acid (AA), a biologically active fatty acid, plays a crucial role as a mediator of signaling pathways involved in cell proliferation and survival. In this study, we investigated the potential role of AA as a proapoptotic agent in CML. Pretreatment of human CML isolated progenitor cells with AA (100 microM for 18 h) induced 71-75% inhibition of in vitro colony formation of granulocyte-macrophage colony-forming units, multilineage colony-forming units, and erythroid burst-forming units. This inhibition was significantly greater than the effect on normal progenitor cells (19-39% growth inhibition of erythroid burst-forming units, multilineage colony-forming units, and granulocyte-macrophage colony-forming units). AA also inhibited growth of the bcr-abl-transformed cell line H7.bcr-abl A54. In contrast, a minimal effect of AA on inhibition of cell growth was observed in the parental nontransformed NSF/N1.H7 cell line. The antiproliferative effect of AA was associated with apoptosis. Gamma-linolenic acid, a precursor of AA, also inhibited cell growth, whereas other unsaturated and saturated fatty acids had no effect. Pharmacological inhibition of cyclooxygenase, lipooxygenase, and cytochrome P450 monooxygenase enzymes prior to exposure to AA did not rescue cells from the inhibitory effect of AA. Moreover, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable arachidonate analogue, also inhibited cell growth, suggesting that the effect of AA did not require further metabolism. Treatment with antioxidants prior to stimulation with AA was also ineffective in preventing its antiproliferative effect. Thus, AA inhibited proliferation of CML cells by inducing apoptotic cell death. The signaling mechanisms of AA-induced inhibition of cell growth appeared to be independent of its conversion into eicosanoids or free radical generation.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácido Araquidónico/farmacología , Células Madre Hematopoyéticas/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Adulto , Animales , Antioxidantes/farmacología , Células de la Médula Ósea/patología , Ácidos Cafeicos/farmacología , División Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/patología , Células Precursoras Eritroides/ultraestructura , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/ultraestructura , Humanos , Indometacina/farmacología , Cetoconazol/farmacología , Masculino , Masoprocol/farmacología , Ratones , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
Activity of the c-jun N-terminal kinase (JNK) has been shown in hematopoietic cells transformed by p210 BCR-ABL. However, analysis has not been reported for hematopoietic cells on the consequences of this activity for c-jun promoter regulation within its distinctive proximal 8-base consensus CRE-like element, an element linked to JNK-mediated increase in c-jun transcription. In the present study, regulation of the proximal c-jun promoter was studied in murine myeloid cells transformed by p210 BCR-ABL. Promoter regulation in p210 BCR-ABL transformed cells was compared with regulation of the promoter in nontransformed interleukin-3 (IL-3)-dependent parental cells. The composition of nuclear AP-1 proteins contained within cells with p210 BCR-ABL, and their binding to the c-jun promoter proximal CRE-like element, was compared with the composition and binding of AP-1 proteins in IL-3-treated parental cells without p210 BCR-ABL. The present analysis found fivefold increased c-jun transcription occurring in p210 BCR-ABL transformed murine myeloid cells possessing a corresponding magnitude of increased kinase activity of JNK, compared with IL-3-stimulated parental cells. Augmented JNK activity was accompanied by increased nuclear abundance of c-jun and c-fos proteins that bound specifically to the proximal c-jun promoter CRE element. Also, representative human leukemic cell lines expressing p210 BCR-ABL and possessing abundant kinase activity of JNK, when compared with parental cells that were deficient in JNK activity, had increased c-jun and c-fos proteins. Finally, to show the relevance of these observations in model systems, we studied blast cells from patients with Philadelphia chromosome-positive acute leukemic transformation, and observed comparable activities of JNK catalysis and c-jun/AP-1 protein relative to the cell lines that possessed p210 BCR-ABL and JNK activity. These studies provide a basis for investigating the set of downstream genes which augmented c-jun/AP-1 activity enlists in the process of transformation by p210 BCR-ABL.
Asunto(s)
Proteínas de Fusión bcr-abl/fisiología , Genes jun , Células Madre Hematopoyéticas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas de Neoplasias/fisiología , Proteínas Quinasas/fisiología , Animales , Transformación Celular Neoplásica , Activación Enzimática , Proteínas de Fusión bcr-abl/genética , Células HL-60 , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , MAP Quinasa Quinasa 4 , Sustancias Macromoleculares , Ratones , Mutagénesis Sitio-Dirigida , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Raised intracellular cyclic AMP (cAMP) has been demonstrated to exert an antiproliferative effect in myeloid cells. How the antiproliferative activity of cAMP is exerted in p210 BCR-ABL transformed myeloid cells was the subject of this investigation. It was hypothesized that cyclin dependent kinase 4, cdk4, might be a critical target enzyme to affect the related events of c-myc transcription and progression through G1 phase of the cell cycle within cells transformed by p210 BCR-ABL, and further, that cdk4 might be downregulated by cAMP to inhibit proliferation. In order to investigate the regulatory role of cdk4, synchronized cells were studied. In p210 BCR-ABL transformed cells transiting early G1 phase, treatment with a cAMP analogue led to inhibition of cyclin D1 synthesis, and marked reduction of cdk4 kinase activity. Within cells in which cdk4 was inhibited by cAMP, there was augmented interaction of E2F1 with the retinoblastoma protein, pRb in a nuclear matrix-associated cell fraction. As a result of E2F1 sequestration, raised intracellular cAMP was found to inhibit c-myc transcription in p210 BCR-ABL transformed myeloid cells synchronously transiting the early G1 phase of the cell cycle. A target of this transcriptional suppression exerted by cAMP was the E2F site of the c-myc P2 promoter. On the other hand, cyclin D1 content was not reduced by cAMP in these cells when it was applied at a later cell cycle stage at the interface between G1 and S. Corresponding to lack of cyclin D1 inhibition in these later G1-to-S phase cells, cdk4 activity was only modestly suppressed, and c-myc mRNA expression was also inhibited to a lesser degree. These studies show that Rb interaction with E2F1 is regulated by cdk4 and cyclin D1 within p210 BCR-ABL transformed leukemia cells in early G1 phase of the cell cycle. In this context, both cyclin D1 and cdk4 are subject to the level of intracellular cAMP. This interaction between Rb and E2F1, which is subject to the level of cAMP, is critical to transcriptional control of c-myc. Further, pRb regulation of E2F activity affects cellular potential for G1-S phase transition in p210 BCR-ABL transformed myeloid cells, in part, via its effect on c-myc transcription.
Asunto(s)
AMP Cíclico/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Fase G1/fisiología , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas , ARN Mensajero/metabolismo , Animales , Línea Celular Transformada , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Ciclina D1 , Quinasa 4 Dependiente de la Ciclina , Proteínas de Fusión bcr-abl , Fase G1/efectos de los fármacos , Luciferasas/metabolismo , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-myc/genética , Fase S/efectos de los fármacos , Fase S/fisiología , Activación TranscripcionalRESUMEN
Steel factor (SLF) acts synergistically with various hematopoietic growth factors that use the Jak-Stat pathways in vivo and in vitro, although the contribution by SLF to this pathway is unknown. We show here that SLF induces time- and dose-dependent phosphorylation of Stat3 in the human growth factor-dependent cell lines MO7e and TF-1. This phosphorylation occurs exclusively on serine residues. Simultaneous stimulation with SLF plus other cytokines that induce tyrosine phosphorylation of Stat3, such as interleukin-9 (IL-9) in MO7e cells or IL-6 in TF-1 cells, resulted in tyrosine phosphorylation and enhanced serine phosphorylation of Stat3. Serine phosphorylation alone did not promote nuclear translocation or DNA binding activity to the sis-inducible element of Stat3. However, costimulation with SLF plus IL-9 in MO7e cells resulted in the nuclear translocation of serine-hyperphosphorylated Stat3. Serine phosphorylation of Stat3 was also observed by the stimulation of cells with granulocyte-macrophage colony-stimulating factor and IL-3, which do not induce tyrosine phosphorylation of Stat3. These results suggest that SLF might modulate the Jak-Stat3 pathway by serine phosphorylation and that the Jak-Stat pathway may be differentially regulated by the combinational stimulation of two or more cytokines.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Fosfoproteínas/biosíntesis , Fosfoserina/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Factor de Células Madre/farmacología , Transactivadores/metabolismo , Secuencia de Bases , Núcleo Celular/metabolismo , Citocinas/farmacología , Factores de Crecimiento de Célula Hematopoyética/fisiología , Humanos , Leucemia Eritroblástica Aguda/patología , Leucemia Megacarioblástica Aguda/patología , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT3 , Transducción de Señal , Células Tumorales CultivadasRESUMEN
It is becoming increasingly clear that the adeno-associated virus 2 (AAV)-based vector system may prove to be useful for high-efficiency gene transfer in human cells as well as for potential gene therapy in humans. A recombinant AAV vector containing the gene for a human hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), was constructed and used to infect COS-1 cells, a monkey kidney cell line. COS-1 cells infected with the recombinant virus, but not mock-infected cells, expressed high levels of the human GM-CSF gene transcripts. Furthermore, in co-cultivation experiments with the recombinant virus-infected cells, but not in those with mock-infected cells, active proliferation of a GM-CSF-dependent human megakaryocytic leukemia cell line, M07e, could be obtained in the absence of exogenously added GM-CSF. The recombinant GM-CSF protein released into the supernatant was biologically active in progenitor cell assays carried out with primary human hematopoietic cells, and this activity was specifically abrogated by treatment of the supernatant with anti-GM-CSF antibodies. This recombinant virus may be potentially useful in the management and gene therapy of a variety of malignant disorders in the human hematopoietic system.
Asunto(s)
Dependovirus/genética , Expresión Génica , Técnicas de Transferencia de Gen , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Animales , Northern Blotting , Southern Blotting , División Celular , Línea Celular , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Haplorrinos , Células Madre Hematopoyéticas/citología , Humanos , Riñón , Leucemia Megacarioblástica Aguda/patología , Proteínas Recombinantes/farmacología , Células Tumorales CultivadasRESUMEN
We have previously shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression induced by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in the murine stromal cell line +/+.1-LDA 11 involves activation of phospholipase A2 (PLA2). Furthermore, induction of GM-CSF gene expression due to release of arachidonic acid as a result of PLA2 activation was mediated by the transcriptional factor c-jun. In the present study, we have investigated the potential mechanism involved in the induction of c-jun gene expression by arachidonic acid. Arachidonic acid induced transcription of c-jun mRNA. Downregulation of protein kinase C (PKC) by chronic exposure of stromal cells to the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 400 nmol/L) did not effect c-jun expression induced by arachidonate. Moreover, pretreatment of cells with the PKC inhibitor, calphostin C (1 mumol/L), caused a marked decrease of c-jun expression induced by TPA, but had no influence on c-jun expression induced by arachidonate. To explore the hypothesis that a tyrosine kinase signalling pathway, independent of PKC activation, was involved in arachidonate-induced c-jun expression, stromal cells were pretreated with the protein tyrosine kinase inhibitor, genistein, before challenge with arachidonic acid. Arachidonate 50 mumol/L)-induced c-jun expression was inhibited, in a dose- and time-dependent manner, by genistein. Genistein similarly inhibited c-jun expression in stromal cells exposed to IL-1 (500 U/mL) plus TNF-alpha (500 U/mL). The potential role of a tyrosine kinase pathway in arachidonate-mediated c-jun expression was further investigated by assaying the tyrosine kinase activity of cells challenged with arachidonic acid, IL-1, and TNF-alpha. Exposure of stromal cells to arachidonic acid induced a 2.1-fold increase in intracellular tyrosine kinase activity determined by phosphorylation of the synthetic peptide, raytide, in the presence of [gamma-32P]-ATP. Similarly, IL-1 and TNF-alpha induced 1.7- and 2.4-fold increases in tyrosine protein kinase activity, respectively. The effect of arachidonic acid on tyrosine kinase activity was inhibited by genistein and was enhanced by sodium vanadate. The increase of protein tyrosine kinase activity detected in arachidonate-stimulated cells was associated, in a dose- and time-dependent fashion, with tyrosine phosphorylation of 240-, 40-, and 29-kD substrates. These results are consistent with the hypothesis that a tyrosine phosphorylation process is triggered by arachidonate as an early event in the signalling pathway that leads to increased expression of c-jun.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Ácido Araquidónico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes jun/efectos de los fármacos , Interleucina-1/farmacología , Proteínas Tirosina Quinasas/fisiología , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Genisteína , Humanos , Isoflavonas/farmacología , Ratones , Naftalenos/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Focal adhesion kinase (p125FAK; FAK) is a protein tyrosine kinase that is tyrosine-phosphorylated in response to v-src-mediated transformation, cell adhesion, and stimulation with neuropeptides. To elucidate a possible functional relationship between FAK and BCR-ABL oncoprotein detected in Philadelphia chromosome-positive (Ph+) leukemias, we investigated the tyrosine phosphorylation state of FAK in a murine growth factor-dependent cell line and in its stable human bcr-abl cDNA transfectant. In interleukin-3 (IL-3)-dependent NFS/N1.H7 cells, tyrosine phosphorylation of FAK was not detected after stimulation with either IL-3 or Steel factor (SLF), both of which involve Ras-mediated signaling pathways. However, stable gene transfection with p210bcr-abl cDNA into H7 cells made these cells growth factor-independent for proliferation and resulted in constitutive tyrosine phosphorylation and kinase activation of FAK. Constitutive phosphorylation and activation of FAK was also observed in all Ph+ leukemia cell lines examined--that is, K562, TS9;22, and YS9;22, which express p210BCR-ABL, and NALM-21 and OM9;22, which express p185BCR-ABL. Ph-negative (Ph-) cell lines, such as MO7e and JM, did not show any detectable tyrosine phosphorylation of FAK. FAK phosphorylation in BCR-ABL-expressing cells was inhibited in a dose-dependent manner by cytochalasin D, a reagent that disrupts the intracellular network of actin filaments. However, no suppression of kinase activity or protein expression of BCR-ABL was observed after treatment with cytochalasin D. A physical association between BCR-ABL and FAK was not apparent. These data suggest that BCR-ABL may be involved in the activation of FAK. Moreover, FAK may be distinct from components in Ras-mediated signaling cascades that are activated by stimulation of myeloid cells with various cytokines.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas de Fusión bcr-abl/metabolismo , Interleucina-3/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Células Madre/farmacología , Animales , División Celular/efectos de los fármacos , Línea Celular , Citocalasina D/farmacología , Activación Enzimática , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Proteínas de Fusión bcr-abl/biosíntesis , Proteínas de Fusión bcr-abl/aislamiento & purificación , Expresión Génica , Humanos , Ratones , Fosforilación , Fosfotirosina/metabolismo , Proteínas Proto-Oncogénicas c-abl/biosíntesis , Proteínas Proto-Oncogénicas c-abl/aislamiento & purificación , Proto-Oncogenes , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Experiments were performed to elucidate the mechanism through which p210 BCR-ABL, by its downstream signals, regulates c-myc messenger RNA expression in hematopoietic cells. We studied a model system in which stable expression of p210 BCR-ABL in interleukin-3 (IL-3) dependent murine myeloid cell lines led to growth factor independent transformation. Active c-myc transcription was observed in p210 BCR-ABL transformed cells by nuclear run-on assay, and in heterologous reporter assays performed with the 5' regulatory region of murine c-myc linked to firefly luciferase. Transcription initiation occurred primarily from the P2 promoter in p210 BCR-ABL transformed cells. Cis and trans elements responsible for transcription initiation from the c-myc P2 promoter were studied. Expression of E2F1 protein in p210 BCR-ABL transformed cells accounted, in part, for binding to the E2F site of the P2 c-myc promoter. The functional importance of E2F1 expression in p210 BCR-ABL transformed cells toward c-myc transcription was established in reporter assays performed with the P2 c-myc promoter containing either wild-type or mutant E2F sites. Mutation of the E2F motif of P2 5' c-myc reduced activity of the promoter by 50%. By gel mobility shift, E2F1 was found in P2 c-myc band shift complexes along with the cyclin-dependent kinase 2. Therefore, coupling of E2F to components of the retinoblastoma-cyclin pathway defines a route from p210 BCR-ABL to c-myc transcription, which is required for p210 BCR-ABL transformation.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Proteínas de Fusión bcr-abl , Genes myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Unión Competitiva , Línea Celular Transformada , ADN/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Genes Reporteros , Luciferasas/metabolismo , Ratones , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1RESUMEN
The intracellular signaling mechanisms that dictate myeloid differentiation and proliferation are discussed. Independent hematopoietic signaling pathways including p21ras pathway, c-myc pathway, and Jak-STAT pathway are defined. Emphasis is given to the process of information integration at the nucleus, by which developmental programs may be converted from binary decisions into the complex response patterns explaining hematopoietic diversity. Coupling between signaling and transcription is emphasized.
Asunto(s)
Células Madre Hematopoyéticas/citología , Transducción de Señal/fisiología , Animales , Diferenciación Celular/fisiología , Ciclinas/fisiología , Factores de Crecimiento de Célula Hematopoyética/fisiología , Humanos , Proteína Oncogénica p21(ras)/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiologíaRESUMEN
Terminal megakaryocytic development is characterized by nuclear poly ploidization, appearance of specific granules, and enhanced expression of membrane platelet glycoproteins. We utilized a human GM-CSF-dependent cell line, MB-02, which is capable of under going megakaryocytic differentiation, to examine the molecular events underlying this process. The responses of MB-02 to the protein kinase C (PKC) agonist, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were examined. GM-CSF dependent proliferation of MB-02, as measured by (3)H-thymidine uptake, was greater than 95% inhibited by TPA (16 nM), but was not affected by the inactive stereoisomer, 4-α-phorbol-12,13-didecanoate (4-αPDD). Transient exposure of cells to GM-CSF after growth factor deprivation led to rapid, high-level expression of normal-sized c-myc mRN A transcripts above baseline. C-myc expression was turned off by TPA (16 nM) stimulation of cells within 2-4 h. This TPA-mediated effect likely occurred at the transcriptional level since the half life of c-myc mRN A induced by GM-CSF was less than 30 min. Treatment of cells with TPA was associated with induction of c-jun and junB mRN A within 1-4 h. The protein products of these transcription factors are known to be part of the transcription factor complex Activator protein 1 (AP-1). Indeed, our data prove a rapid induction of AP-1 protein after TPA stimulation, as shown by mobility shift assays. In addition, TPA treatment resulted in expression of platelet surface glycoprotein IIb/IIIa complex (gpIIb/IIIa). These studies suggest a link between PKC stimulation by TPA and AP-1 activation with downregulation of c-myc transcription on a molecular level. At the cellular level, PKC activation was related to the acquisition of several features of the megakaryocyte development programme associated with the switch from cell proliferation to maturation.
RESUMEN
We have reported modulation, by cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) and by hormonal cyclic-adenosine-monophosphate (cAMP) agonists, of hematopoietic growth factor production in the murine marrow adherent cell line +/+(-)1.LDA11. Previously, we reported that increased intracellular cAMP levels inhibited bioactive granulocyte-macrophage colony-stimulatory factor (GM-CSF) production stimulated by IL-1 or by the synergistic stimulus of IL-1 plus TNF-alpha. On the other hand, increased intracellular cAMP stimulated IL-6 synthesis in +/+(-)1.LDA11 cells. In addition, cAMP was additive with either IL-1 or IL-1 plus TNF-alpha in inducing production of soluble IL-6. In the present study, these observations were pursued mechanistically at the level of messenger RNA (mRNA) production. Northern blot analysis of steady-state mRNA for GM-CSF revealed induction by treatment of +/+(-)1.LDA11 cells with IL-1 or with TNF-alpha. The combined stimulation by IL-1 plus TNF-alpha resulted in supra-additive increases in GM-CSF expression by +/+(-)1.LDA11. Addition to stromal cells of the soluble cAMP agonist 8-bromo-cAMP (8BrcAMP) at 0.5 to 1 mM stimulated IL-6 mRNA expression acting alone, and it was additive with IL-1 or IL-1 plus TNF-alpha in stimulating IL-6 expression. On the other hand, 8BrcAMP inhibited GM-CSF mRNA expression stimulated by IL-1 or IL-1 plus TNF-alpha. Inhibition of GM-CSF mRNA by 8BrcAMP was time-dependent, starting 120 to 180 minutes posttreatment. In addition, inhibition of GM-CSF transcript expression in +/+(-)1.LDA11 by 8BrcAMP required the expression of a labile protein. Nuclear run-on assays revealed that GM-CSF and IL-6 genes were transcriptionally induced in +/+(-)1.LDA11 by incubation with IL-1 plus TNF-alpha. IL-6 transcription was further enhanced by 8BrcAMP co-incubation. More sensitive experiments using a luciferase reporter vector containing the GM-CSF promoter region were necessary to convincingly establish the role of TNF-alpha and 8BrcAMP on transcriptional induction of the GM-CSF gene in +/+(-)1.LDA11 stromal cells. Considering these results and an effect of 8BrcAMP on decreasing GM-CSF transcript stability in actinomycin-D (act-D) decay experiments, we conclude that the inhibitory effect of 8BrcAMP on GM-CSF expression is exerted at the posttranscriptional level. These data demonstrate that the intracellular level of cAMP has an important discriminatory role on expression of the cytokines GM-CSF and IL-6 in a model stromal cell line.
Asunto(s)
Células de la Médula Ósea , Médula Ósea/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Procesamiento Proteico-Postraduccional , Transcripción Genética , Animales , Northern Blotting , Médula Ósea/metabolismo , Línea Celular , AMP Cíclico/análisis , AMP Cíclico/metabolismo , AMP Cíclico/fisiología , Regulación hacia Abajo/efectos de los fármacos , Regulación de la Expresión Génica , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-1/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-6/fisiología , Ratones , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The p210 bcr-abl fusion protein tyrosine kinase oncogene has been implicated in the pathogenesis of chronic granulocytic leukemia (CGL). Specific intracellular functions performed by p210 bcr-abl have recently been delineated. We considered the possibility that p210 bcr-abl may also regulate the abundance of inosine 5'-monophosphate dehydrogenase (IMPDH) which is a rate-limiting enzyme for de novo guanylate synthesis. We performed studies of the inhibition of IMPDH by tiazofurin, which acts as a competitive inhibitor through its active species that mimics nicotinamide adenine dinucleotide (NAD), i.e. thiazole-4-carboxamide adenine dinucleotide (TAD). The mean inhibitory concentration (IC50) of tiazofurin for cellular proliferation inhibition was 2.3-2.8-fold greater in cells expressing p210 bcr-abl than in their corresponding parent cells proliferating under the influence of growth factors or in growth factor-independent derivative cells not expressing detectable p210 bcr-abl. IMPDH activity was 1.5-2.3-fold greater within cells expressing p210 bcr-abl than in their parent cells. This increase in enzyme activity was a result of 2-fold increased IMPDH protein as determined by immunoblotting. In addition, an increase in the Km value for NAD utilization by IMPDH was observed in p210 bcr-abl transformed cells, but this increase was within the range of resident NAD concentrations observed in the cells. Increased IMPDH protein in p210 bcr-abl transformed cells was traced to an increased level of IMP dehydrogenase II messenger RNA. Thus, regulation of IMPDH gene expression is mediated at least in part by the bcr-abl gene product and may therefore be indicative of a specific mechanism of intrinsic resistance to tiazofurin.
Asunto(s)
Resistencia a Medicamentos/genética , Proteínas de Fusión bcr-abl/metabolismo , Expresión Génica , IMP Deshidrogenasa/biosíntesis , Oncogenes , Ribavirina/análogos & derivados , Animales , Antineoplásicos/toxicidad , Northern Blotting , División Celular/efectos de los fármacos , Línea Celular Transformada , Replicación del ADN/efectos de los fármacos , Proteínas de Fusión bcr-abl/biosíntesis , IMP Deshidrogenasa/metabolismo , Ratones , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosfotirosina , Poli A/análisis , Poli A/biosíntesis , ARN/análisis , ARN/biosíntesis , ARN Mensajero , Ribavirina/toxicidad , Timidina/metabolismo , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
Signal transduction pathways evoked by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) to stimulate expression of other cytokines in mesenchymal cells are not clearly understood. Stimulation of the murine bone marrow stromal cell line +/(+)-1.LDA 11 with IL-1 (500 U/ml) in combination with TNF-alpha (500 U/ml) (IL-1 plus TNF-alpha) induced expression of c-jun mRNA as well as granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA. We investigated the possibility that arachidonic acid metabolites, acting through protein kinase C (PKC) and perhaps also through the PKC-responsive transcription factor c-jun/AP-1, may be responsible for regulating GM-CSF transcription in these stromal cells. Expression of GM-CSF mRNA was preceded by IL-1 plus TNF-alpha induced arachidonate release (assayed using the 3H-derivative). Pretreatment of cells with the phospholipase A2 inhibitor quinacrine (20 microM) inhibited accumulation of both c-jun and GM-CSF mRNA but had no influence on expression of other genes induced by IL-1 and TNF-alpha, including leukemia inhibitory factor (LIF). In addition, quinacrine partially blocked IL-1 plus TNF-alpha induced 3H-arachidonic acid release from prelabeled stromal cells. Furthermore, exogenous arachidonate (10 to 50 microM) induced expression of c-jun. To investigate the role of arachidonate in GM-CSF transcription, we used a reporter vector consisting of the murine GM-CSF promoter linked to firefly luciferase. Transfection efficiency was monitored by assessing expression of a constitutively active gene, RSV-beta galactosidase. In this system, quinacrine significantly inhibited IL-1 plus TNF-alpha induced GM-CSF transcription assayed with the reporter construct. Exogenous arachidonic acid alone (10 microM) increased activity of GM-CSF reporter vector 1.5-fold over control. These results are consistent with the hypothesis that arachidonate metabolites are involved in the signaling pathway that leads to IL-1 plus TNF-alpha induced GM-CSF gene expression. Thus, transcriptional activation of GM-CSF gene is mediated, in part, by the arachidonate cascade.