RESUMEN
BACKGROUND: Severe congenital neutropenia (SCN) is an immunodeficiency characterized by disturbed myelopoiesis and an absolute neutrophil count (ANC) <0.5 × 10(9)/L. SCN is also a premalignant condition; a significant proportion of patients develop myelodysplastic syndrome or leukemia (MDS/L). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for SCN. PROCEDURE: Since 2004, eight HSCT have been performed in seven patients at our center. The indications were transformation to MDS/L (n = 2), granulocyte colony-stimulating factor receptor (CSF3R) mutation(s) (n = 2), granulocyte colony-stimulating factor (G-CSF) resistance (n = 2), and at the patient's own request (n = 1). RESULTS: The mean age at transplantation was 13 years (2.8-28 years) (mean follow-up 32 months, range 21-60). Three patients harbored ELANE mutations, three HAX1 mutations, and in one patient no causative mutation was identified. Two of the ELANE mutations were novel mutations. Three patients initially received myeloablative conditioning and four had reduced intensity conditioning (RIC). Three grafts were from HLA-identical siblings, three from matched unrelated donors and two were cord blood units. Engraftment occurred in all patients. Two of seven (29%) patients died; both had MDS/L and both were among the three that underwent myeloablative conditioning. One patient has chronic GVHD 2 years post-transplant. CONCLUSIONS: The role of HSCT should be explored further in patients with SCN. In particular, the influence of the conditioning regime needs to be evaluated in a larger cohort of patients.
Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre Hematopoyéticas , Acondicionamiento Pretrasplante , Adolescente , Adulto , Niño , Preescolar , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Femenino , Humanos , Leucemia/etiología , Leucemia/terapia , Masculino , Síndromes Mielodisplásicos/etiología , Síndromes Mielodisplásicos/terapia , Neutropenia/congénito , Neutropenia/terapia , Tasa de Supervivencia , Resultado del Tratamiento , Adulto JovenRESUMEN
In approximately 20% of cases of severe congenital neutropenia (SCN), mutations are found in the gene encoding the granulocyte colony-stimulating factor receptor (G-CSF-R). These mutations introduce premature stop codons, which result in truncation of 82-98 COOH-terminal amino acids of the receptor. SCN patients who develop secondary myelodysplastic syndrome and acute myeloid leukemia almost invariably acquired a GCSFR mutation, suggesting that this genetic alteration represents a key step in leukemogenesis. Here we show that an equivalent mutation targeted in mice (gcsfr-Delta715) results in the selective expansion of the G-CSF- responsive progenitor (G-CFC) compartment in the bone marrow. In addition, in vivo treatment of gcsfr-Delta715 mice with G-CSF results in increased production of neutrophils leading to a sustained neutrophilia. This hyperproliferative response to G-CSF is accompanied by prolonged activation of signal transducer and activator of transcription (STAT) complexes and extended cell surface expression of mutant receptors due to defective internalization. In view of the continuous G-CSF treatment of SCN patients, these data provide insight into why progenitor cells expressing truncated receptors clonally expand in vivo, and why these cells may be targets for additional genetic events leading to leukemia.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/farmacología , Síndromes de Inmunodeficiencia/inmunología , Leucemia Mieloide/genética , Activación de Linfocitos/efectos de los fármacos , Proteínas de la Leche , Neutropenia/genética , Receptores de Factor Estimulante de Colonias de Granulocito/deficiencia , Enfermedad Aguda , Animales , Médula Ósea/patología , Diferenciación Celular/genética , División Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Ensayo de Unidades Formadoras de Colonias , Contraindicaciones , Proteínas de Unión al ADN/metabolismo , Endocitosis , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Células Madre Hematopoyéticas/patología , Humanos , Síndromes de Inmunodeficiencia/genética , Cinética , Leucemia Mieloide/inducido químicamente , Ratones , Ratones Transgénicos , Neutropenia/congénito , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Receptores de Factor Estimulante de Colonias de Granulocito/fisiología , Factor de Transcripción STAT1 , Factor de Transcripción STAT5 , Eliminación de Secuencia , Organismos Libres de Patógenos Específicos , Transactivadores/metabolismoRESUMEN
Severe congenital neutropenia (SCN) is a heterogeneous condition characterized by a drastic reduction in circulating neutrophils and a maturation arrest of myeloid progenitor cells in the bone marrow. Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF). Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment. When this mutant G-CSF-R was expressed in myeloid cells, it was defective in both proliferation and survival signaling. This correlated with diminished activation of the receptor complex as determined by signal transducer and activator of transcription (STAT) activation, although activation of STAT5 was more affected than STAT3. Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor. This suggests that the mutation in the extracellular domain affects ligand-receptor complex formation with severe consequences for intracellular signal transduction. Together these data add to our understanding of the mechanisms of cytokine receptor signaling, emphasize the role of GCSFR mutations in the etiology of SCN, and implicate such mutations in G-CSF hyporesponsiveness.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Neutropenia/congénito , Neutropenia/tratamiento farmacológico , Mutación Puntual , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Células de la Médula Ósea/metabolismo , División Celular , Relación Dosis-Respuesta a Droga , Femenino , Factor Estimulante de Colonias de Granulocitos/metabolismo , Humanos , Lactante , Ligandos , Modelos Moleculares , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transducción de SeñalRESUMEN
Signals induced by granulocyte colony-stimulating factor (G-CSF), the major cytokine involved in neutrophil development, are tightly controlled by ligand-induced receptor internalization. Truncated G-CSF receptors (G-CSF-Rs) that fail to internalize show sustained proliferation and defective differentiation signaling. Steady-state forward routing also determines cell surface levels of cytokine receptors, but mechanisms controlling this are poorly understood. Here, we show that WD40 and suppressor of cytokine signaling (SOCS) box protein-2 (Wsb-2), an SOCS box-containing WD40 protein with currently unknown function, binds to the COOH-terminal region of G-CSF-R. Removal of this region did not affect internalization, yet resulted in increased membrane expression of G-CSF-R and enhanced proliferation signaling at the expense of differentiation induction. Conversely, Wsb-2 binding to the G-CSF-R reduced its cell surface expression and inhibited proliferation signaling. These effects depended on the SOCS box involved in ubiquitylation and on cytosolic lysines of G-CSF-R and imply a major role for ubiquitylation through the G-CSF-R C-terminus in forward routing of the receptor. Importantly, the Wsb-2 gene is commonly disrupted by virus integrations in mouse leukemia. We conclude that control of forward routing of G-CSF-R is essential for a balanced response of myeloid progenitors to G-CSF and suggest that disturbance of this balance may contribute to myeloid leukemia.
Asunto(s)
Proteínas Portadoras/metabolismo , Factor Estimulante de Colonias de Granulocitos/metabolismo , Leucemia Mieloide/etiología , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Diferenciación Celular , Membrana Celular/química , Membrana Celular/metabolismo , Proliferación Celular , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Ratones , Mapeo de Interacción de Proteínas , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/análisis , Proteínas Supresoras de la Señalización de Citocinas/genética , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismoRESUMEN
Tumor necrosis factor (TNF) inhibits granulocyte-colony-stimulating factor (G-CSF)-induced human acute myeloid leukemia (AML) growth in vitro. Incubation of blasts from three patients with AML in serum-free medium with TNF (10(3) U/ml), and subsequent binding studies using 125I-G-CSF reveal that TNF downregulates the numbers of G-CSF receptors by approximately 70%. G-CSF receptor numbers on purified blood granulocytes are also downmodulated by TNF. Downregulation of G-CSF receptor expression becomes evident within 10 min after incubation of the cells with TNF at 37 degrees C and is not associated with an apparent change of the dissociation constant (Kd). The TNF effect does not occur at 0 degrees C and cannot be induced by IL-2, IL-6, or GM-CSF. TNF probably exerts its effect through activation of protein kinase C (PKC) as the TNF effect on G-CSF receptor levels can be mimicked by 12-O-tetradecanoylphorbol-13- acetate. The PKC inhibitor Staurosporine (Sigma Chemical Co., St. Louis, MO) as well as protease inhibitors can completely prevent G-CSF receptor downmodulation. Thus, it appears TNF may act as a regulator of G-CSF receptor expression in myeloid cells and shut off G-CSF dependent hematopoiesis. The regulatory ability of TNF may explain the antagonism between TNF and G-CSF stimulation.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/metabolismo , Granulocitos/fisiología , Leucemia Mieloide/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Factor de Necrosis Tumoral alfa/farmacología , Enfermedad Aguda , Alcaloides/farmacología , Regulación hacia Abajo/efectos de los fármacos , Granulocitos/efectos de los fármacos , Humanos , Técnicas In Vitro , Inhibidores de Proteasas/farmacología , Estaurosporina , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
The granulocyte colony-stimulating factor receptor (G-CSF-R) transduces signals important for the proliferation and maturation of myeloid progenitor cells. To identify functionally important regions in the cytoplasmic domain of the G-CSF-R, we compared the actions of the wild-type receptor, two mutants, and a natural splice variant in transfectants of the mouse pro-B cell line BAF3 and two myeloid cell lines, 32D and L-GM. A region of 55 amino acids adjacent to the transmembrane domain was found to be sufficient for generating a growth signal. The immediate downstream sequence of 30 amino acids substantially enhanced the growth signaling in the three cell lines. In contrast, the carboxy-terminal part of 98 amino acids strongly inhibited growth signaling in the two myeloid cell lines but not in BAF3 cells. Truncation of this region lead to an inability of the G-CSF-R to transduce maturation signals in L-GM cells. An alternative carboxy tail present in a splice variant of the G-CSF-R also inhibited growth signaling, notably in both the myeloid cells and BAF3 cells, but appeared not to be involved in maturation.
Asunto(s)
Diferenciación Celular/fisiología , División Celular/fisiología , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular/genética , División Celular/genética , Línea Celular , Citoplasma/metabolismo , ADN Complementario/genética , Variación Genética , Granulocitos/citología , Granulocitos/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Transducción de Señal , TransfecciónRESUMEN
Mutations in the granulocyte colony-stimulating factor receptor (G-CSF-R) gene leading to a truncated protein have been identified in a cohort of neutropenia patients highly predisposed to acute myeloid leukemia. Such mutations act in a dominant manner resulting in hyperproliferation but impaired differentiation in response to G-CSF. This is due, at least in part, to defective internalization and loss of binding sites for several negative regulators, leading to sustained receptor activation. However, those signaling pathways responsible for mediating the hyperproliferative function have remained unclear. In this study, analysis of an additional G-CSF-R mutant confirmed the importance of residues downstream of Box 2 as important contributors to the sustained proliferation. However, maximal proliferation correlated with the ability to robustly activate signal transducer and activator of transcription (STAT) 5 in a sustained manner, whereas co-expression of dominant-negative STAT5, but not dominant-negative STAT3, was able to inhibit G-CSF-stimulated proliferation from a truncated receptor. Furthermore, a Janus kinase (JAK) inhibitor also strongly reduced the proliferative response, whereas inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) or phosphatidylinositol (PI) 3-kinase reduced proliferation to a lesser degree. These data suggest that sustained JAK2/STAT5 activation is a major contributor to the hyperproliferative function of truncated G-CSF receptors, with pathways involving MEK and PI 3-kinase playing a reduced role.
Asunto(s)
Proliferación Celular , Mutación , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Transducción de Señal/fisiología , Animales , Línea Celular Tumoral , Janus Quinasa 2/fisiología , Sistema de Señalización de MAP Quinasas , Ratones , Proteína Oncogénica p21(ras)/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Factor de Transcripción STAT3/fisiología , Factor de Transcripción STAT5/fisiologíaRESUMEN
Hematopoiesis, the process of blood cell formation, is orchestrated by cytokines and growth factors that stimulate the expansion of different progenitor cell subsets and regulate their survival and differentiation into mature blood cells. Granulocyte colony-stimulating factor (G-CSF) is the major hematopoietic growth factor involved in the control of neutrophil development. G-CSF is now applied on a routine basis in the clinic for treatment of congenital and acquired neutropenias. G-CSF activates a receptor of the hematopoietin receptor superfamily, the G-CSF receptor (G-CSF-R), which subsequently triggers multiple signaling mechanisms. Here we review how these mechanisms contribute to the specific responses of hematopoietic cells to G-CSF and how perturbations in the function of the G-CSF-R are implicated in various types of myeloid disease.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/fisiología , Enfermedades Hematológicas/etiología , Hematopoyesis/fisiología , Receptores de Factor Estimulante de Colonias de Granulocito/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Endocitosis , Retroalimentación , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/farmacología , Hematopoyesis/genética , Humanos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Proteínas Recombinantes , Transducción de SeñalRESUMEN
The signal transducer and activator of transcription (STAT) proteins have been implicated in cytokine-regulated proliferation, differentiation and cell survival. Granulocyte colony-stimulating factor (G-CSF), a regulator of granulocytic differentiation, induces a robust and sustained activation of STAT3. Here, we show that introduction of dominant negative (DN) forms of STAT3 interferes with G-CSF-induced differentiation and survival in murine 32D cells. G-CSF induces expression of the cyclin-dependent kinase (cdk) inhibitor p27(KiP1) (but not p21(CiP1)), which is completely blocked by DN-STAT3. The ability of tyrosine-to-phenylalanine substitution mutants of the G-CSF receptor to activate STAT3 strongly correlated with their capacity to induce p27 expression and their ability to mediate differentiation and survival, suggesting a causal relationship between STAT3 activation, p27 expression and the observed cellular responses. We identified a putative STAT binding site in the promoter region of p27 that showed both STAT3 binding in electrophoretic mobility shift assays and functional activity in luciferase reporter assays. Finally, we studied G-CSF-induced responses in primary bone marrow and spleen cells of p27-deficient mice. Compared with wild-type, myeloid progenitors from p27-deficient mice showed significantly increased proliferation and reduced differentiation in response to G-CSF. These findings indicate that STAT3 controls myeloid differentiation, at least partly, via upregulation of p27(Kip1).
Asunto(s)
Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos , Factor Estimulante de Colonias de Granulocitos/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Transactivadores/metabolismo , Proteínas Supresoras de Tumor , Animales , Sitios de Unión , Diferenciación Celular , División Celular , Supervivencia Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN/genética , Expresión Génica , Factor Estimulante de Colonias de Granulocitos/genética , Células HeLa , Humanos , Ligandos , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Mutagénesis , Neutrófilos/citología , Regiones Promotoras Genéticas , Factor de Transcripción STAT3 , Células Madre/citología , Transactivadores/genética , Transfección , Células Tumorales CultivadasRESUMEN
Approximately 25-30% of childhood pre-B cell acute lymphoblastic leukemias (pre-B ALL) is characterized by the presence of a (1;19)(q23;p13.3) translocation. The presence of this translocation is generally accompanied by a poor prognosis. The chimeric gene resulting from this chromosomal rearrangement encodes a hybrid transcription factor, E2A-Pbx1. In an attempt to delineate the genetic cascade initiated by E2A-Pbx1, we sought to identify genes that are deregulated by this transcription factor in t(1;19) pre-B ALL. We show here that the gene encoding the granulocyte colony-stimulating factor receptor (G-CSFr) is specifically upregulated in pre-B cells expressing E2A-Pbx1. G-CSFr is also expressed in cell lines established from t(1;19) pre-B cell leukemia and on primary t(1;19) tumor cells, but not on control cells. These data indicate that G-CSFr gene is a target for deregulation by E2A-Pbx1.
Asunto(s)
Linfoma de Burkitt/genética , Cromosomas Humanos Par 19 , Cromosomas Humanos Par 1 , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Translocación Genética , Linfocitos B , Linfoma de Burkitt/metabolismo , Células Madre Hematopoyéticas , Proteínas de Homeodominio/genética , Humanos , Proteínas de Fusión Oncogénica/genética , Receptores de Factor Estimulante de Colonias de Granulocito/fisiología , Células Tumorales CultivadasRESUMEN
In vitro colony-forming abilities of highly purified primitive hematopoietic cells in eight cases of myelodysplastic syndrome (MDS) were studied using the blast cell colony assay. Blast cell colony formation from seven normal bone marrow (NBM) samples was studied in parallel. Blast cell colonies were formed in 7/8 cases of MDS, the numbers not significantly differing from those generated by NBM. In contrast the more mature hematopoietic progenitors (granulocyte-erythroid-macrophage-megakaryocyte colony-forming unit, CFU-GEMM; erythroid burst-forming units, BFU-E; granulocyte colony-forming units, CFU-G; eosinophilic colony-forming units, CFU-Eo) were severely depressed in numbers in MDS marrow. After replating of blast cell colonies in secondary cultures, colonies were obtained in 5/8 MDS cases. A marked difference was evident in the composition of the secondary colonies between MDS and normal marrow. Whereas secondary colonies derived from normal blast cell colonies consisted of about 45% of erythroid cells, MDS blast colonies generated mainly colonies of the granulocytic-monocytic lineage and no erythroid colonies. The normal quantitative level of CFU-blast progenitors in MDS in the context of their impaired ability to generate lineage specific progeny upon secondary plating suggests that the incompetence of maturation of MDS may reside in the CFU-blast progenitor cell being incapable of properly responding to growth factor stimulation.
Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/patología , Síndromes Mielodisplásicos/fisiopatología , Adulto , Anciano , Médula Ósea/patología , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Células Precursoras Eritroides/patología , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/patologíaRESUMEN
Previous studies have shown that the phenotypes of progenitors of human AML (AML-CFU) are variable, reflecting arrests at different stages of maturation. We were interested to seek discrepancies between the surface properties of AML precursors and normal bone marrow colony formers in order to detect minimal numbers of AML cells among normal bone marrow cells in remission bone marrow. Therefore, we selected two surface markers, the MoAb CD34, reactive with blast cells, and Vim-2, a surface marker reactive with mature myeloid cells, and determined the antigen density of these markers (relative fluorescence intensity using fluorescence-activated cell sorting) for normal marrow and AML progenitors. While these markers defined an identical phenotype (CD34++/Vim-2-/+) for a broad spectrum of normal progenitors, i.e., CFU-GEMM, BFU-e, day 15 CFU-GM, and day 7 CFU-GM, referred to as the "normal" progenitor phenotype, AML progenitors frequently exhibited different phenotypes. In 12 of 20 cases the phenotypes of the majority of AML progenitors were discrepant from the normal surface profile, i.e., according to one marker in 8 cases (CD34-/+/Vim-2-/+ or CD34++/Vim-2++) and two markers in 4 cases (CD34-/+/Vim-2++). Since these data indicate that AML and normal progenitors were frequently distinguishable, we then determined the potential utility of these phenotypic dissimilarities for detection of minimal disease. Artificial mixtures of normal bone marrow and minimal numbers (0.1-1%) of AML cells were prepared. Based upon the phenotypic discrepancies, AML metaphases were successfully demonstrated in these mixtures following cell sorting and culture. Thus, it appears that minimal numbers of AML mitoses can be identified with an approximate 10(-2) to 10(-3) sensitivity by taking advantage of differential coexpression of surface antigens.
Asunto(s)
Examen de la Médula Ósea/métodos , Médula Ósea/patología , Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/patología , Monitorización Inmunológica , Células Madre Neoplásicas/patología , Anticuerpos Monoclonales , Antígenos de Neoplasias/análisis , Diferenciación Celular , Separación Celular , Citometría de Flujo , Células Madre Hematopoyéticas/clasificación , Humanos , FenotipoRESUMEN
Receptors of most hematopoietic growth factors are structurally related and grouped in the hematopoietin or cytokine receptor superfamily. In this paper, we will first review the general principles of hematopoietin receptor complex formation and cytoplasmic signaling. Subsequently, the significance of defective hematopoietic growth factor receptors for the development of hematological diseases will be discussed.
Asunto(s)
Sustancias de Crecimiento/química , Hematopoyesis , Receptores del Factor Estimulante de Colonias/química , Secuencia de Aminoácidos , Animales , Humanos , Ligandos , Datos de Secuencia Molecular , Agregación de Receptores , Transducción de SeñalRESUMEN
Interleukin-1 (IL-1) has hemopoietin-1 (H-1) activity, i.e., it synergizes with macrophage-colony stimulating factor (M-CSF), granulocyte-macrophage-CSF (GM-CSF) and interleukin-3 (IL-3) in stimulating in vitro colony formation of hematopoietic progenitor cells. In this study the synergistic activity of IL-1 was investigated on IL-3 and GM-CSF induced growth of acute myeloid leukemia colony forming cells (AML-CFU) in vitro. Among 12 cases of human AML, IL-1 significantly elevated IL-3 stimulated colony numbers in eight instances and enhanced GM-CSF induced colony growth in five cases. As IL-1 is an inducer of cytokine production and since tumor necrosis factor (TNF) elevates IL-3 or GM-CSF induced proliferation of AML-CFU, we examined whether IL-1 enhanced AML-CFU growth via the induction of TNF production. Neutralizing anti-TNF-alpha antibodies significantly decreased IL-1/IL-3 or IL-1/GM-CSF stimulated colony numbers in six of seven cases studied, whereas anti-TNF-beta had no effect, indicating that endogenously produced TNF-alpha costimulated the growth of AML-CFU. Furthermore, AML blast cells stimulated by IL-1 released increased amounts of TNF-alpha (between 25 and 533 pg/ml; median 255 pg/ml) into the culture medium (TNF-alpha specific radioimmunoassay) as compared with noninduced AML cells (less than 1 to 149 pg TNF-alpha/ml; median 31 pg/ml). Thus, the effect of IL-1 on AML-CFU proliferation is not the result of direct activation of AML progenitors, but IL-1 stimulates the release of TNF-alpha by AML cells and endogenous TNF subsequently synergizes with IL-3 or GM-CSF.
Asunto(s)
Factores Estimulantes de Colonias/farmacología , Sustancias de Crecimiento/farmacología , Interleucina-1/farmacología , Interleucina-3/farmacología , Leucemia Mieloide Aguda/patología , Factor de Necrosis Tumoral alfa/biosíntesis , Anticuerpos , División Celular , Sinergismo Farmacológico , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Factores de Crecimiento de Célula Hematopoyética , Humanos , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Ensayo de Tumor de Célula MadreRESUMEN
We have tested a panel of recombinant hematopoietic growth factors (HGF) including the interleukins (IL) 1, 2, 3, 4, and 6 and the colony stimulating factors GM-CSF, G-CSF, M-CSF for their ability to induce proliferation of precursor B acute lymphoblastic leukemia cells (ALL) from 19 patients. In Ficoll-Isopaque isolated and T cell-depleted ALL bone marrow samples, IL2 (two cases), IL3 (four cases), and GM-CSF (one case) infrequently stimulated DNA synthesis measured by 3H-thymidine (TdR) uptake, and the other recombinant growth factors completely failed to do so. In repeat experiments with ALL blasts purified by fluorescence activated cell sorting (FACS), IL2, IL3, and GM-CSF responses could not be reproduced, suggesting that nonleukemic contaminant cells, and not the ALL blasts, had been stimulated by these factors. Cocktails containing combinations of IL1-IL4 and IL6 also lacked proliferation inducing potency. Depending on the purity of the incubated ALL cell samples, an impure preparation of B cell growth factors that has been reported to contain a highly effective stimulatory activity for precursor B ALL cells induced proliferation of residual normal cells as well as the ALL cells, as was evident from combined analysis of DNA synthesis and karyotyping. Exposure of the ALL blasts to artificial activators of protein kinase C and Ca2+ mobilization resulted in significant rises in 3H-TdR uptake, suggesting that these intracellular compounds are involved in transducing signals that upregulate proliferation. Although it remains possible that some of the human recombinant growth factors promote the growth of precursor B ALL cells in combination with other stimuli, a dominant role in the regulation of proliferation of these cells cannot be attributed to any of these cytokines at the present time.
Asunto(s)
Factores Estimulantes de Colonias/farmacología , Interleucinas/farmacología , Leucemia Linfoide/patología , Células Madre Neoplásicas/patología , Linfocitos B , Calcimicina/farmacología , División Celular/efectos de los fármacos , Humanos , Interleucina-4 , Proteínas Recombinantes/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
In this study we demonstrate that tumor necrosis factors (TNF alpha and TNF beta) are potent modulators of the in vitro proliferation of human AML cells. Blast cells from 11 cases of acute myeloblastic leukemia (AML) were incubated with recombinant TNF alpha or TNF beta in serum-free 3H-TdR uptake and colony culture systems in the presence or absence of recombinant interleukin-3 (IL-3), granulocyte macrophage colony-stimulating factor (GM-CSF), G-CSF, or M-CSF. Depending on the supplemented CSF, TNF could upregulate or suppress AML blast proliferation. Enhancement of AML growth by TNF was observed in the presence of IL-3 (in 9 of 11 cases in 3H-TdR assay; 6 of 9 cases in colony assay) and GM-CSF (in 8 of 11 cases in 3H-TdR assay; 4 of 9 cases in colony assay). In certain cases in which IL-3 or GM-CSF alone was unable to induce proliferative responses of AML cells, the simultaneous addition of TNF elicited colony growth and DNA synthesis suggesting a synergistic action between TNF and IL-3 or GM-CSF. In contrast, TNF suppressed G-CSF-induced growth (9 of 10 cases in 3H-TdR assay; 5 of 6 cases in colony assay). TNF could also stimulate DNA synthesis (in 2 of 11 cases) or colony formation (in 2 of 9 cases) in AML cultures without the addition of other growth factors. Experiments with neutralizing antibodies and specific radioimmunoassays for individual CSFs showed that the synergistic and antagonistic effects of TNF on AML growth could not be attributed to a release of one of these CSFs by the AML cells. The opposing consequence of exposure of AML blasts to TNF are of interest in view of our understanding of the pathophysiology of AML growth and the in vivo application of recombinant cytokines in AML patients.
Asunto(s)
Factores Estimulantes de Colonias/farmacología , Leucemia Mieloide Aguda/patología , Factor de Necrosis Tumoral alfa/farmacología , Adulto , División Celular/efectos de los fármacos , Factores Estimulantes de Colonias/biosíntesis , Factor Estimulante de Colonias de Granulocitos , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Sustancias de Crecimiento/farmacología , Humanos , Interleucina-3/farmacología , Células Tumorales CultivadasRESUMEN
To assess cooperation between G-CSF signals and C/EBPalpha, we characterized Ba/F3 pro-B cell lines expressing C/EBPalphaWT-ER and the G-CSF receptor (GCSFR). In these lines, GCSFR signals can be evaluated independent of their effect on C/EBPalpha levels. G-CSF alone did not induce the MPO, NE, LF, or PU.1 RNAs, and C/EBPalphaWT-ER alone stimulated low-level MPO and high-level PU.1 expression. Simultaneous activation of the GCSFR and C/EBPalphaWT-ER markedly increased MPO and NE induction at 24 h, and LF mRNA was detected at 48 h. G-CSF did not increase endogenous GCSFR, endogenous C/EBPalpha or exogenous C/EBPalphaWT-ER levels, and C/EBPalphaWT-ER did not induce endogenous or exogenous GCSFR. Several GCSFR mutants were also co-expressed with C/EBPalphaYWT-ER. Mutation of all four cytoplasmic tyrosines prevented NE induction but enhanced MPO induction. Mutation of Y704 was required for increased MPO induction. Consistent with this finding, removing IL-3 without G-CSF addition enabled MPO, but not NE, induction by C/EBPalphaWT-ER. GCSFR signals or related signals from other receptors may cooperate with C/EBPalpha to direct differentiation of normal myeloid stem cells.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/fisiología , Regulación Enzimológica de la Expresión Génica , Elastasa de Leucocito/genética , Peroxidasa/genética , Receptores de Factor Estimulante de Colonias de Granulocito/fisiología , Animales , División Celular , Línea Celular , Factor Estimulante de Colonias de Granulocitos/farmacología , Humanos , Ratones , TransgenesRESUMEN
The development of mature granulocytes from hematopoietic precursor cells is controlled by a myriad of transcription factors which regulate the expression of essential genes, including those encoding growth factors and their receptors, enzymes, adhesion molecules, and transcription factors themselves. In particular, C/EBPalpha, PU.1, CBF, and c-Myb have emerged as critical players during early granulopoiesis. These transcription factors interact with one another as well as other factors to regulate the expression of a variety of genes important in granulocytic lineage commitment. An important goal remains to understand in greater detail how these various factors act in concert with signals emanating from cytokine receptors to influence the various steps of maturation, from the pluripotent hematopoietic stem cell, to a committed myeloid progenitor, to myeloid precursors, and ultimately to mature granulocytes.
Asunto(s)
Citocinas/fisiología , Granulocitos/citología , Leucopoyesis/fisiología , Transducción de Señal , Factores de Transcripción/fisiología , Citocinas/metabolismo , Humanos , Factores de Transcripción/metabolismoRESUMEN
In the summer of 1997, the first meeting on 'Acute Leukemias with Structurally Altered Core Binding Factor Subunits' was held. During the meeting, which attracted more than 140 participants, many recognized experts in the field of CBF and leukemia were present. In this short report we summarize new data on CBF involvement in leukemia and other diseases that were presented during the meeting.
Asunto(s)
Inversión Cromosómica , Cromosomas Humanos Par 12 , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 21 , Cromosomas Humanos Par 8 , Proteínas de Unión al ADN/genética , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Factores de Transcripción/genética , Translocación Genética , Factores de Unión al Sitio Principal , HumanosRESUMEN
Among 52 patients diagnosed as acute myeloid leukemia (AML), nine cases were found in which interleukin-5 (IL-5) induced a proliferative response in the leukemic cells, as measured by the stimulation of DNA synthesis or colony formation in vitro. All cases (n = 7) with the cytogenetic abnormality t(8;21)(q22;q22) belonged to this group of IL-5 responders. Of the additional two cases, one had an apparently normal karyotype, but the other expressed a dicentric chromosome 21, an abnormality also involving the breakpoint region 21q22. The leukemic cells of the IL-5 responsive patients could also be stimulated to proliferate by IL-3, GM-CSF and G-CSF, and in some cases by IL-6 or M-CSF. Immunophenotypic analysis revealed the presence of the immature hematopoietic cell antigen CD34, the myelomonocytic maturation antigens CD13 and CD33, in association with the B-cell related surface marker CD19 on the leukemic cells. Immunoglobulin mu and T-cell receptor beta-genes in the leukemic cells were in germline configuration. Upon incubation in colony culture, clonogenic cells were capable of producing progeny showing eosinophilic or neutrophilic maturation following stimulation with IL-5 or G-CSF, respectively. It is concluded that IL-5 responsive AML represents a subgroup of leukemia with distinct immunotypic and cytogenetic features.