RESUMEN
Fms-like tyrosine kinase 3 (FLT3) is expressed in hematopoietic progenitor cells. An internal tandem duplication (ITD) of FLT3 (FLT3/ITD) is the most frequent mutation in human adult acute myeloid leukemia (AML). FLT3/ITD contributes to the constitutive activation of FLT3 itself and its downstream signal components, mitogen-activated protein kinase and signal transducers and activators of transcription 5 (STAT5), and enables interleukin (IL)-3-dependent cell lines to grow autonomously. In the present study, we showed the specific association of FLT3/ITD with Lyn, which led to the phosphorylation of Lyn in vivo. We also demonstrated that FLT3/ITD receptors displayed a higher affinity to bind to Lyn than wild-type FLT3 receptors in vitro and that this affinity was relative to the intensity of tyrosil phosphorylation of the receptor. Both treatment with small interfering RNA (siRNA) targeting Lyn and the Src family kinase inhibitor PP2 suppressed the IL-3-independent growth of FLT3/ITD-expressing 32D cells (FLT3/ITD-32D), reducing the constitutive phosphorylation of Lyn and STAT5. PP2 treatment of mice transplanted with FLT3/ITD-32D cells blocked the onset of tumors and decreased the size of established tumors. These results demonstrate that Lyn is an important component of the signal transduction pathway specific to FLT3/ITD and can be a therapeutic target in the treatment of AML with FLT3/ITD.
Asunto(s)
Leucemia Mieloide/enzimología , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/metabolismo , Familia-src Quinasas/metabolismo , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular/efectos de los fármacos , Línea Celular/trasplante , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/genética , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/genética , Proteínas de Neoplasias/genética , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/fisiología , Factor de Transcripción STAT5/metabolismo , Secuencias Repetidas en Tándem , Transfección , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genética , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
The hematopoietically expressed GATA family of transcription factors function as key regulators of blood cell fate. Among these, GATA-2 is implicated in the survival and growth of multipotential progenitors. Here we report that the promyelocytic leukemia protein (PML) can complex with GATA-2 and potentiate its transactivation capacity. The binding is mediated through interaction of the zinc finger region of GATA-2 and the B-box domain of PML. The B-box region of PML is retained in the PML-RARalpha (retinoic acid receptor alpha) fusion protein generated by the t(15;17) translocation characteristic of acute promyelocytic leukemia (APL). Consistent with this, we provide evidence that GATA-2 can physically associate with PML-RARalpha. Functional experiments further demonstrated that this interaction has the capacity to render GATA-dependent transcription inducible by retinoic acid, raising the possibility that GATA target genes may be involved in the molecular pathogenesis of APL.
Asunto(s)
Cromosomas Humanos Par 15 , Cromosomas Humanos Par 17 , Proteínas de Unión al ADN/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Proteínas de Fusión Oncogénica , Receptores de Ácido Retinoico/metabolismo , Factores de Transcripción/metabolismo , Translocación Genética , Animales , Células COS , Línea Celular , Núcleo Celular/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/química , Factor de Transcripción GATA2 , Humanos , Leucemia Promielocítica Aguda/metabolismo , Ratones , Proteínas de Neoplasias/química , Plásmidos/metabolismo , Pruebas de Precipitina , Proteína de la Leucemia Promielocítica , Unión Proteica , Estructura Terciaria de Proteína , Receptor alfa de Ácido Retinoico , Factores de Transcripción/química , Activación Transcripcional , Tretinoina/farmacología , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Técnicas del Sistema de Dos HíbridosRESUMEN
A novel human myeloid leukemia cell line, NKM-1, was established from a patient with acute myeloid leukemia (FAB classification M2). The cells were positive for myeloperoxidase staining and cluster of differentiation 15 cell surface antigen. Radiolabeled recombinant human granulocyte (G) colony-stimulating factor (CSF) was used, and 60 specific binding sites/cell with a Kd 100 pmol/liter were demonstrated on the cell surface. 125I-G-CSF binding was not inhibited by interleukin-3, granulocyte-macrophage CSF, or macrophage (M) CSF. NKM-1 cells also expressed M-CSF receptors detected by c-fms mRNA expression. In concordance with the receptor expression, NKM-1 cells proliferated in response to exogenous G-CSF or M-CSF in a dose-dependent manner (0.1-100 ng/ml), while interleukin-3 or granulocyte-macrophage CSF had no effect. Colony-forming capacity of NKM-1 cells in semisolid agar was also enhanced with the addition of 10 ng/ml of G-CSF or M-CSF but decreased at higher concentrations. During CSF stimulation, no remarkable changes were observed morphologically and phenotypically. The stimulatory effect of G-CSF and M-CSF on the cell growth was additive. Neither G-CSF-binding capacity nor c-fms mRNA expression was altered by pretreatment with M-CSF or G-CSF, respectively. This cell line may provide a useful in vitro model for the study of CSF roles in myeloid leukemia cell proliferation.
Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Receptores de Factor Estimulante de Colonias de Granulocito/biosíntesis , Adulto , Antígenos de Superficie/análisis , Northern Blotting , División Celular/efectos de los fármacos , Líquido Cefalorraquídeo/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-3/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/inmunología , Factor Estimulante de Colonias de Macrófagos/fisiología , Masculino , ARN/análisis , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Granulocyte colony-stimulating factor (G-CSF) enhanced surface transferrin receptor (TfR) expression in two human myeloid leukemia cell lines, NKM-1 and NOMO-1, which possess G-CSF receptors. Radioligand-binding assay revealed that 10 ng/ml G-CSF significantly increased TfR to 186 +/- 20 and 276 +/- 38% of control for NKM-1 cells and NOMO-1 cells, respectively, in a 24-h culture. Scatchard analysis showed the increase of transferrin (Tf)-binding sites but no change in the receptor affinity. The enhanced TfR expression was not mediated either by the kinetic change of receptor cycling or by cellular iron content. Immunoprecipitation with anti-TfR antibody was used, and the increased biosynthesis of the receptor was demonstrated in G-CSF-stimulated cells. Northern blot analysis showed a 2- to 3-fold increase of TfR mRNA of NKM-1 cells cultured in medium containing Tf and G-CSF, whereas the mRNA declined without G-CSF. The effect of G-CSF on the TfR mRNA was observed within 2 h, which preceded the increase of surface TfR and the transition to the S phase of the cell cycle. G-CSF also potentiated TfR expression in freshly obtained myeloid leukemia cells. The present study shows up-regulation of TfR expression by G-CSF in myeloid leukemia cells and provides evidence that the regulation is mediated by controlling the steady-state level of the mRNA.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Leucemia Mieloide/metabolismo , Receptores de Transferrina/genética , Regulación hacia Arriba , Transporte Biológico , Línea Celular , Replicación del ADN/efectos de los fármacos , Humanos , Cinética , Leucemia Megacarioblástica Aguda/metabolismo , Receptores de Transferrina/efectos de los fármacos , Receptores de Transferrina/metabolismo , Timidina/metabolismo , Transferrina/metabolismoRESUMEN
The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) on DNA topoisomerase II (topo II) expression was studied in two human acute myelogenous leukemia cell lines, NKM-1 and NOMO-1, which express G-CSF receptor and proliferate in response to exogenous G-CSF. Northern blot analysis revealed that the level of topo II mRNA in 16-h stimulated cells in serum-free medium with G-CSF (10 ng/ml) was approximately 2-fold higher than that in cells without G-CSF. Enhanced topo II mRNA expression was detectable within 3 h after the addition of G-CSF. Topo II activity in crude nuclear extracts from 16-h G-CSF-stimulated cells was also found to be approximately 2-fold greater than that from unstimulated cells. According to in vitro cytotoxic assay, the sensitivity of G-CSF-stimulated cells to intercalating (daunorubicin) and nonintercalating (etoposide) topo II-targeting drugs increased significantly, whereas no enhancement of sensitivity was observed with an alkylating agent (4-hydroperoxycyclophosphamide). The augmented drug sensitivity observed was not due to the increased level of drug transport, as suggested by the similar extent of [3H]etoposide uptake between G-CSF-stimulated and unstimulated cells. By measuring the topo II mRNA and the cytotoxicity of the above mentioned drugs, we obtained essentially the same results in G-CSF-responsive leukemia cells isolated from three acute myeloblastic leukemia patients, as observed in the cultured cell lines. These findings strongly suggest that the sensitivity to "topo II-targeting drugs" could be augmented by exogenous G-CSF through elevated topo II activity in G-CSF-responsive leukemia cells.
Asunto(s)
ADN-Topoisomerasas de Tipo II/genética , Factor Estimulante de Colonias de Granulocitos/farmacología , Northern Blotting , Ciclofosfamida/análogos & derivados , Ciclofosfamida/toxicidad , ADN-Topoisomerasas de Tipo II/metabolismo , Daunorrubicina/toxicidad , Relación Dosis-Respuesta a Droga , Etopósido/metabolismo , Etopósido/toxicidad , Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Proteínas Recombinantes , Células Tumorales CultivadasRESUMEN
We have recently identified an internal tandem duplication of the human Flt3 gene in approximately 20% of acute myeloid leukemia (AML) cases. In the present study, the wild-type and the mutant Flt3 genes were transfected into two IL-3-dependent cell lines, 32D and BA/F3 cells. Mutant Flt3-transfected cells exhibited autonomous growth while wild-type Flt3-transfected cells with the continuous stimulation of Flt3 ligand exhibited a minimal proliferation. Cells expressing mutant Flt3 showed constitutive activation of STAT5 and MAP kinase. In contrast, Flt3 ligand stimulation caused rapid activation of MAP kinase but not STAT5 in cells expressing wild-type Flt3. Finally, we found constitutive activation of MAP kinase and STAT5 in all clinical samples of AML patients with mutant Flt3. Our study shows the significance of internal tandem duplication of Flt3 receptors for leukemia cell expansion.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Duplicación de Gen , Interleucina-3/fisiología , Proteínas de la Leche , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Muerte Celular/efectos de los fármacos , División Celular/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Inhibidores de Crecimiento/farmacología , Humanos , Leucemia Eritroblástica Aguda/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Datos de Secuencia Molecular , Mutación/genética , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Secuencias Repetitivas de Aminoácido , Factor de Transcripción STAT5 , Células Tumorales Cultivadas , Tirosina Quinasa 3 Similar a fmsRESUMEN
Transcription factor c-Myb plays important roles in cell survival and differentiation in immature hematopoietic cells. Here we demonstrate that c-Myb is acetylated at the carboxyl-terminal conserved domain by histone acetyltransferase p300 both in vitro and in vivo. The acetylation sites in vivo have been located at the lysine residues of the conserved domain (K471, K480, K485) by the use of the mutant Myb (Myb-KAmut), in which all three lysine residues are substituted into alanine. Electrophoretic mobility shift assay reveals that Myb-KAmut shows higher DNA binding activity than wild type c-Myb and that acetylation of c-Myb in vitro by p300 causes dramatic increase in DNA binding activity. Accordingly, transactivation activity of both mim-1 and CD34 promoters by Myb-KAmut is higher than that driven by wild type c-Myb. Furthermore, the bromodomain of p300, in addition to the histone acetyltransferase (HAT) domain, is required for effective acetylation of c-Myb, and hGCN5 is revealed to be a factor acetyl-transferase for c-Myb in vitro. We present a new manner of post-translational modification of the c-Myb protein and the potential significance of the acetylation in c-Myb.
Asunto(s)
Acetiltransferasas/fisiología , Proteínas de Ciclo Celular/fisiología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores/fisiología , Acetilación , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , ADN/metabolismo , Histona Acetiltransferasas , Humanos , Datos de Secuencia Molecular , Conejos , Factores de Transcripción , Factores de Transcripción p300-CBPRESUMEN
Mitogen-activated protein (MAP) kinase appears to be one of the key regulators of cell proliferation and differentiation. Very little, however, has been revealed as to how MAP kinase is involved in leukemogenesis. We have studied the activation of the MAP kinase pathway in 100 human primary leukemia cells including 73 acute myelogenous leukemias (AMLs). Forty acute leukemia samples (40% of the total), including 37 AML samples (51% of AML), showed activation of MAP kinase as revealed by the mobility shift of the phosphorylated form of the protein and by in vitro kinase assay. This activation was correlated with MAP kinase kinase activity in these cells. In contrast, none of 14 chronic myelogenous leukemia samples showed the activation of MAP kinase. These results suggest that the MAP kinase pathway is constitutively activated in a subset of primary acute leukemias, and thus indicate the possible role of the constitutively activated MAP kinase in leukemogenesis.
Asunto(s)
Genes ras/fisiología , Leucemia/enzimología , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Activación Enzimática , Humanos , Leucemia Mielomonocítica Aguda/enzimología , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Fosforilación , Proteínas Quinasas/genética , Células Tumorales CultivadasRESUMEN
An internal tandem duplication (ITD) of the FLT3 gene is found in nearly 20% of acute myeloid leukemia (AML) and 5% of myelodysplastic syndrome cases. Our serial studies on 51 samples with the FLT3 gene mutation indicated that the ITD was frequently (47/51) clustered in the tyrosine-rich stretch from codon 589 to 599 and rarely (3/51) in its downstream region, both of which are located within the juxtamembrane (JM) domain. One remaining sample had an insertion into the JM domain of nucleotides of unknown origin. To elucidate the biological relevance of the ITD or the insertion, we expressed various types of mutant FLT3 in Cos 7 cells. All mutant FLT3 studied were ligand-independently dimerized and their tyrosine residues were phosphorylated. The Y589 of FLT3 was essential for the phosphorylation in the wild FLT3, but a Y589F conversion did not affect the phosphorylation status of the mutant FLT3. These findings suggest that the elongation of the JM domain rather than increase of tyrosine residues causes gain-of-function of FLT3. Thus, ITD is a novel modality of somatic mutation which activates its product. Since the DNA corresponding to codon 593 to 602 potentially forms a palindromic intermediate, we propose that a DNA-replication error might be associated with generating the ITD of the FLT3 gene.
Asunto(s)
Leucemia Mieloide/genética , Mutación/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Enfermedad Aguda , Animales , Células COS/metabolismo , Mapeo Cromosómico , Humanos , Leucemia Mieloide/metabolismo , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Análisis de Secuencia , Tirosina Quinasa 3 Similar a fmsRESUMEN
We previously reported the fusion of the TEL gene to the Syk gene in myelodysplastic syndrome with t(9;12)(q22;p12). TEL-Syk fusion transformed interleukin-3 (IL-3)-dependent murine hematopoietic cell line BaF3 to growth factor independence. Here, we investigate the intracellular signal transduction of the stable transfectants. TEL-Syk fusion protein was associated with the p85 subunit of phosphatidyl inositol 3 kinase (PI3-K) followed by the activation of Akt in the absence of IL-3. Vav, phospholipase C-gamma2 and mitogen-activated protein kinase (MAPK) were also constitutively activated. TEL-Syk also activated the signal transducer and activator of transcription 5 (STAT5) in the absence of Janus kinase 2 activation. None of these kinases were phosphorylated in the BaF3 cells transfected with TELDeltaPNT-Syk in which the oligomerization domain of TEL was deleted. Inhibitor analysis showed that the MAPK pathway was important in TEL-Syk-mediated cell proliferation. The immunofluorescence technique revealed that the TEL-Syk fusion protein was located in the cytoplasm. These data suggest that TEL-Syk fusion protein in the cytoplasm leads to the constitutive activation of PI3-K/Akt, MAPK and STAT5 signal pathways, which are closely involved in IL-3-independent cell proliferation of BaF3 cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas de la Leche , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Sustitución de Aminoácidos , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Interleucina-3/farmacología , Péptidos y Proteínas de Señalización Intracelular , Janus Quinasa 2 , Cinética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-ets , Proteínas Recombinantes de Fusión , Factor de Transcripción STAT5 , Quinasa Syk , Transfección , Proteína ETS de Variante de Translocación 6RESUMEN
Somatic mutation of the FLT3 gene, in which the juxtamembrane domain has an internal tandem duplication, is found in 20% of human acute myeloid leukemias and causes constitutive tyrosine phosphorylation of the products. In this study, we observed that the transfection of mutant FLT3 gene into an IL3-dependent murine cell line, 32D, abrogated the IL3-dependency. Subcutaneous injection of the transformed 32D cells caused leukemia in addition to subcutaneous tumors in C3H/HeJ mice. To develop a FLT3-targeted therapy, we examined tyrosine kinase inhibitors for in vitro growth suppression of the transformed 32D cells. A tyrosine kinase inhibitor, herbimycin A, remarkably inhibited the growth of the transformed 32D cells at 0.1 microM, at which concentration it was ineffective in parental 32D cells. Herbimycin A suppressed the constitutive tyrosine phosphorylation of the mutant FLT3 but not the phosphorylation of the ligand-stimulated wild-type FLT3. In mice transplanted with the transformed 32D cells, the administration of herbimycin A prolonged the latency of disease or completely prevented leukemia, depending on the number of cells inoculated and schedule of drug administration. These results suggest that mutant FLT3 is a promising target for tyrosine kinase inhibitors in the treatment of leukemia.
Asunto(s)
Antineoplásicos/uso terapéutico , Transformación Celular Neoplásica/genética , Inhibidores Enzimáticos/uso terapéutico , Leucemia Experimental/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Animales , Benzoquinonas , Línea Celular Transformada/trasplante , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Genisteína/uso terapéutico , Humanos , Hidroquinonas/uso terapéutico , Interleucina-3/farmacología , Lactamas Macrocíclicas , Ratones , Ratones Endogámicos C3H , Trasplante de Neoplasias , Fosforilación/efectos de los fármacos , Ftalimidas/uso terapéutico , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Quinonas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Rifabutina/análogos & derivados , Transducción de Señal/efectos de los fármacos , Transfección , Tirfostinos/uso terapéutico , Tirosina Quinasa 3 Similar a fmsRESUMEN
We investigated the effect of the histone deacetylase inhibitors (HDIs), trichostatin A and trapoxin A on leukemia cells and cell lines from the viewpoint of differentiation induction. TSA induced differentiation in erythroid cell lines by itself, whereas it synergistically enhanced the differentiation that was directed by all-trans retinoic acid (ATRA) or vitamin D3 in U937, HL60 and NB4 cells. The combined treatment of HDI with ATRA induced differentiation in ATRA-resistant HL60 and NB4 cells. The transcriptional expression during the treatment with HDI was examined in HL60, U937 and MEG-O1. Cell cycle-regulator genes (p21waf1 and p16INK4A) were upregulated or constantly expressed, erythroid-specific genes (GATA-1, beta-globin) were silent or downregulated, and housekeeping genes (beta-actin and GAPDH) were constantly expressed. Twelve of 35 (34%) clinical samples from AML patients ranging from M0 to M7 also displayed both phenotypical and morphological changes by the treatment with TSA alone. HDIs are thus the potent inducer or enhancer of differentiation in acute myeloid leukemia and regulate transcription in an ordered manner.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Leucemia Mieloide/tratamiento farmacológico , Péptidos , Células 3T3 , Enfermedad Aguda , Animales , Antibacterianos/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Precursoras Eritroides/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ácidos Hidroxámicos/uso terapéutico , Megacariocitos/efectos de los fármacos , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Mitogen-activated protein (MAP) kinases act as transducers of extracellular signaling via tyrosine kinase-growth factor receptors and G-protein-linked receptors to transcription factors. Constitutive activation of MAP kinase has been observed in a variety of solid tumors including renal cancer and breast cancer. Recently, we have reported that constitutively activated MAP kinase was observed in 50% of human primary acute myeloid leukemia (AML) cells. Ras is one of the components of G-proteins and transduces the signal from cytokine receptors to raf-1 theoretically resulting in the activation of MAP kinase pathway. In the present study, we have examined the correlation of Ras mutations and the activation of MAP kinase pathway in patients with AML. Twenty out of 22 AML cases with activating N-Ras mutations showed no phosphorylated forms of ERK2. ERK2 phosphorylation was tightly correlated with ERK1 phosphorylation and MAP kinase activity detected by in vitro kinase assay. Three samples with N-Ras mutations were stimulated with IL-3, GM-CSF and G-CSF separately but ERK2 activation was induced in none of these samples stimulated with these cytokines. In contrast, ERK2 was constitutively activated in all of four pancreatic carcinoma cases with K-Ras mutation at codon 12. These results suggest that function of the Ras mutations may be different between solid tumors, such as pancreatic carcinoma and colorectal carcinoma, and AML. Mutated Ras does not always stimulate MAP kinase pathway constitutively and may rather inhibit classical MAP kinase cascade in AML blasts from leukemia patients.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Genes ras , Leucemia Mieloide/patología , Proteínas Quinasas Activadas por Mitógenos , Proteínas de Neoplasias/metabolismo , Mutación Puntual , Procesamiento Proteico-Postraduccional , Transducción de Señal/fisiología , Enfermedad Aguda , Carcinoma/enzimología , Carcinoma/genética , Carcinoma/patología , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Factores de Crecimiento de Célula Hematopoyética/farmacología , Humanos , Leucemia Mieloide/enzimología , Leucemia Mieloide/genética , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Fosforilación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Point mutations of the transcription factor AML1 are associated with leukemogenesis in acute myeloblastic leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and mutations in the second tyrosine kinase domain of the Fms-like tyrosine kinase 3 (FLT3) gene represent the most frequent genetic alterations in AML. However, such mutations per se appear to be insufficient for leukemic transformation. To evaluate whether both AML1 and FLT3 mutations contribute to leukemogenesis, we analyzed mutations of these genes in AML M0 subtype in whom AML1 mutations were predominantly observed. Of 51 patients, eight showed a mutation in the Runt domain of the AML1 gene: one heterozygous missense mutation with normal function, five heterozygous frameshift mutations and two biallelic nonsense or frameshift mutations, resulting in haploinsufficiency or complete loss of the AML1 activities. On the other hand, a total of 10 of 49 patients examined had the FLT3 mutation. We detected the FLT3 mutation in five of eight (63%) patients with AML1 mutation, whereas five of 41 (12%) without AML1 mutation showed the FLT3 mutation (P=0.0055). These observations suggest that reduced AML1 activities predispose cells to the acquisition of the activating FLT3 mutation as a secondary event leading to full transformation in AML M0.
Asunto(s)
Proteínas de Unión al ADN/genética , Mutación del Sistema de Lectura , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Factores de Transcripción/genética , Transformación Celular Neoplásica/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Regulación Leucémica de la Expresión Génica , Humanos , Polimorfismo Conformacional Retorcido-Simple , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirosina Quinasa 3 Similar a fmsRESUMEN
There are few molecular biologic determinants that are prognostic for patients with acute myeloid leukemia (AML). Hence, we examined whether cellular levels of the cyclin-dependent kinase inhibitor p27Kip1 in acute myeloid leukemia could be used to predict clinical outcome in AML. Using immunoblot analysis, levels of p27 were assessed in blast cells from 72 AML patients who were registered and treated by the identical chemotherapy protocol. AML cases were classified into three groups on the basis of the percentage of the expression level of p27 compared to a control cell line. AML cases exhibiting p27 expression at low, moderate, and high levels were 43, 9, and 20 cases, respectively. No significant differences in the rates of complete remission (CR) were observed among the three groups. Although the level of p27 expression was not correlated with any other possible prognostic markers, such as age, white blood cell count, chromosome abnormalities, and FAB subclasses, patients with high p27 expression had a significantly increased disease-free survival (DFS) (78% vs 19%, P = 0.004). We further examined the expression of cyclin E at the protein level in all 72 AML cases. We observed a statistically significant correlation between a high cyclin E level and a high p27 level (P < 0.005). However, we failed to find any correlation between the rates of CR or DFS and cyclin E expression. The present study reveals that levels of p27 expression can be one of the useful prognostic molecular markers for AML. Leukemia (2000) 14, 28-33.
Asunto(s)
Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Leucemia Mieloide/fisiopatología , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas Supresoras de Tumor , Enfermedad Aguda , Adolescente , Adulto , Anciano , Biomarcadores de Tumor , Ciclina E/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , PronósticoRESUMEN
OBJECTIVES: To study the role of the hematopoietic transcription factor GATA-2 in long terminal repeat (LTR)-directed transcriptional activation of HIV-1 in hematopoietic progenitor cells, and to investigate possible GATA-2 binding sites in HIV-1 LTR. DESIGN AND METHODS: Wild-type HIV-1 LTR, or mutants, ligated to a luciferase reporter gene with or without a GATA-2 expression vector, were transfected into COS cells, and standardized luciferase activity was examined. The binding activity of GATA-2 to these sites was examined by electrophoretic mobility shift assay. These wild-type or mutant reporter genes were also transfected into the murine hematopoietic progenitor cells, BAF3, in which GATA-2 was the predominantly expressed transcription factor of the GATA family, to assay LTR-directed transcription in intact hematopoietic machinery. Using a Tat expression plasmid for cotransfection, the influence of Tat protein on GATA-2-induced transactivation was determined. RESULTS: In COS cells, LTR-dependent transactivation was highly enhanced by the coexpression of GATA-2. Experiments with mutant LTR suggested the presence of multiple GATA-2 binding sites, of which the major sites were identified. Cotransfection of Tat with GATA-2 indicated that GATA-2 and Tat synergistically enhanced the transcriptional activity. Transfection experiments in BAF3 cells showed that the disruption of these GATA sites diminished LTR-driven activity to 40% of the wild-type. CONCLUSIONS: GATA-2 may be a key host cell regulator of HIV-1 expression in hematopoietic stem cells. Manipulating this transactivation may represent a valuable approach to controlling virus production in infected hematopoietic progenitors. To elucidate the possible interaction between GATA-2 and Tat protein in vivo might give new insights to the mechanism of impaired hematopoiesis in AIDS patients.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/fisiología , Células Madre Hematopoyéticas/virología , Factores de Transcripción/fisiología , Replicación Viral/fisiología , Animales , Células COS , Factor de Transcripción GATA2 , Humanos , Activación TranscripcionalRESUMEN
The transcriptional activity of transcription factors is regulated by phosphorylation. The uncontrolled expression and constitutive activation of transcriptional regulators have been reported to cause malignant diseases. However, little is known about the phosphorylation status of tissue-specific transcription factors in human primary malignancies. Here we present the first insights into both protein expression and phosphorylation of transcription factors in a large-scale study of patients with acute myeloid leukemia (AML). We examined the expression and phosphorylation status of hematopoietic transcription factors PU.1 and C/EBP beta detected by the retarded mobility of the phosphorylated forms of the proteins. The rate of protein expression differed among French-American-British (FAB) subclasses. The expression of C/EBP beta and PU.1 were detectable in 77% and 61%, respectively, of 90 AML samples examined. The expressed PU.1 and C/EBP beta was always accompanied with both phosphorylated and unphosphorylated forms of PU.1 and C/EBP beta, respectively. Statistical significance was observed between PU.1 expression (phosphorylation) and FAB classification (M0, M4, or M5 versus M2 or M3, P < .0001). PU.1 and C/EBP beta were simultaneously detected in all M0, M4, M5 and peripheral blood monocytes, whereas in M2 and M3, the expression of the 2 transcription factors varied among samples. Examination of protein expression and phosphorylation of these lineage-specific molecules may help us to understand the functional characteristics of AML.
Asunto(s)
Crisis Blástica , Proteínas de Unión al ADN/metabolismo , Hematopoyesis , Leucemia Mieloide/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Enfermedad Aguda , Médula Ósea/química , Médula Ósea/patología , Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/fisiología , Humanos , Japón/epidemiología , Leucemia Mieloide/epidemiología , Leucemia Mieloide/fisiopatología , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/fisiología , Fosforilación , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/biosíntesis , Transactivadores/fisiología , Factores de Transcripción/biosíntesis , Factores de Transcripción/fisiologíaRESUMEN
Selective neutropenia lasting over five months occurred in a 17-year-old adolescent male who received an allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling for severe aplastic anemia. Bone marrow specimens showed maturation arrest of myeloid precursor cells despite sustained engraftment. Cytogenetic analyses revealed complete donor-type chimerism in hematopoietic cells and mixed lymphoid chimerism. The patient received a second BMT from the same donor following more intensive conditioning, including total body irradiation. Neutrophil recovery was rapid and complete donor-type hematopoietic and lymphoid chimerism was observed within three weeks of the second transplant. The present case suggests that prolonged selective neutropenia following BMT is due to residual host-derived immunity which is resistant to the standard immunosuppressive conditioning used prior to BMT for aplastic anemia.
Asunto(s)
Trasplante de Médula Ósea/efectos adversos , Transfusión de Linfocitos , Neutropenia/etiología , Neutrófilos/inmunología , Donantes de Tejidos , Adolescente , Enfermedad Crónica , Terapia Combinada , Humanos , Masculino , Trasplante HomólogoRESUMEN
We recently identified several constitutively active forms of signal transducers and activators of transcription 5 (STAT5) using polymerase chain reaction-driven random mutagenesis followed by retrovirus-mediated expression screening. All constitutively active STAT5 showed constitutive phosphorylation on their tyrosine residues and induced factor-independent growth in a mouse interleukin-3-dependent cell line, Ba/F3. Sequence analysis of these active STAT5 revealed two important mutations: S710F and N642H. The N642H mutation localized in the SH2 domain was able to induce autonomous growth of Ba/F3 cells by itself, whereas S710F in the effector domain was able to induce autonomous growth of Ba/F3 cells in concert with a second mutation including H298R and E150G. Recently, constitutive activation of STAT5 has been reported in patients' leukemic cells and is implicated in leukemogenesis. We attempted to clarify whether leukemic cells harbored activating mutations primarily in STAT5 proteins, and analyzed the sequence of STAT5 derived from 49 leukemic patients. No mutations were found, however, in the regions surrounding S710 and N642 of STAT5A and corresponding residues of STAT5B. We also cloned full-length cDNAs for STAT5s from three patients whose leukemic cells exhibited constitutive tyrosine phosphorylation of the STAT5 protein and expressed the derived STAT5 proteins in Ba/F3 cells. However, none of these clones exhibited constitutive tyrosine phosphorylation or gave rise to FI proliferation of Ba/F3 cells. These results indicate that constitutive activation of STAT5 is a secondary event in most leukemias.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de la Leche , Transactivadores/genética , Animales , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/farmacología , Humanos , Leucemia/sangre , Leucemia/etiología , Leucemia/genética , Ratones , Mutación , Factor de Transcripción STAT5 , Transactivadores/aislamiento & purificación , Transactivadores/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Proteínas Supresoras de TumorRESUMEN
We present an 18-year-old woman who was diagnosed with acute myeloblastic leukemia (AML M2), and in whom chromosome analysis of bone marrow cells revealed t(7;11), an abnormality rarely found in leukemias with a differentiation potency. She relapsed 1 year after complete remission was achieved by chemotherapy. Bone marrow examination then revealed a t(7;11) abnormality in 48 of 50 metaphases examined, even when there were less than 7.5% leukemic blasts in the marrow, indicating that the morphologically normal cells were derived from leukemic blasts. The number of leukemia clones with the additional abnormalities in chromosome 5 increased, with concurrent development of eosinophilia, fever, asthma-like symptoms, erythema, itching, and hepatosplenomegaly. Elevation of interleukin 5 (IL-5) in serum and an enhanced expression of IL-5 mRNA were also detected. The increase in IL-5 may have been produced by an abnormality on chromosome 5.