RESUMEN
Models of macrophage subtypes require molecular characterization to reliably facilitate their differentiation. Although CD16+ (Fc-gamma III receptor) monocytes that express CD163 (a hemoglobin/haptoglobin receptor) have been implicated in a variety of disease states, the conditions necessary to establish lineages of these cell subtypes remains unknown. The current investigations utilize a cell line derived from acute myelogenous leukemia lineage, MonoMac-1, to interrogate the factors that promote the development of CD16+ macrophages that express CD163. Results implicate the glucocorticoid pathway as well as c-fms signaling based on the action of dexamethasone and macrophage colony-stimulating factor-1 in promoting CD16+ expression, in addition to phorbol myristate acetate and lipopolysaccharides treatment. The ability of glucocorticoid and c-fms receptor antagonists to inhibit CD16+ cell formation further establishes the role of these pathways in CD16 expression in this cell line. In view of the inherent difficulty in working with primary cells as well as donor variation, cell lines may be preferable to primary cells for their consistency. We envision that the process we use to induce CD16 expression in this cell type will be useful for screening and identification of drug candidates potentially useful for the treatment of diseases where the etiology involves the expansion of the CD16+ monocytes subset or the accumulation of CD163+ tissue macrophages.
Asunto(s)
Diferenciación Celular/fisiología , Glucocorticoides/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Transducción de Señal/fisiología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/fisiología , Genes fms/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de IgG/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The proliferation, differentiation and survival of mononuclear phagocytes depend on signals from the receptor for macrophage colony-stimulating factor, CSF1R. The mammalian Csf1r locus contains a highly conserved super-enhancer, the fms-intronic regulatory element (FIRE). Here we show that genomic deletion of FIRE in mice selectively impacts CSF1R expression and tissue macrophage development in specific tissues. Deletion of FIRE ablates macrophage development from murine embryonic stem cells. Csf1rΔFIRE/ΔFIRE mice lack macrophages in the embryo, brain microglia and resident macrophages in the skin, kidney, heart and peritoneum. The homeostasis of other macrophage populations and monocytes is unaffected, but monocytes and their progenitors in bone marrow lack surface CSF1R. Finally, Csf1rΔFIRE/ΔFIRE mice are healthy and fertile without the growth, neurological or developmental abnormalities reported in Csf1r-/- rodents. Csf1rΔFIRE/ΔFIRE mice thus provide a model to explore the homeostatic, physiological and immunological functions of tissue-specific macrophage populations in adult animals.
Asunto(s)
Genes fms/genética , Macrófagos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Eliminación de Secuencia , Animales , Secuencia de Bases , Diferenciación Celular , Proliferación Celular , Modelos Animales de Enfermedad , Células Madre Embrionarias/patología , Factor de Crecimiento Epidérmico , Femenino , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Macrófagos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Monocitos/metabolismo , Fagocitosis , Células RAW 264.7 , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
OBJECTIVE: To explore the pathogenesis of idiopathic thrombocytopenic purpura (ITP) and improve the differential diagnosis from myelodysplastic syndromes (MDS). METHODS: Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was performed to detect the point mutation of codon 12,13 in N-ras gene and codon 301, 969 in fms gene in adult and aged ITP and MDS patients. RESULTS: In 25 ITP patients, N-ras mutation and fms mutation were detected in one each (4%). Mutations were found in 3 of 8 MDS patients: two (25%) with N-ras mutation and one (12.5%) with fms mutation. CONCLUSIONS: Patients with N-ras or fms gene mutation diagnosed as MDS rather than ITP.
Asunto(s)
Genes fms/genética , Genes ras/genética , Síndromes Mielodisplásicos/genética , Púrpura Trombocitopénica Idiopática/genética , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación Puntual , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Microglia play a critical role in neurodegenerative diseases and in the brain aging process. Yet, little is known about the functional dynamics of microglia during aging. Thus, using young and aging transgenic mice expressing enhanced-green fluorescent protein (EGFP) under the promoter of the c-fms gene for macrophage-colony stimulating factor receptor, we evaluated in vivo-induced inflammatory responses of EGFP-expressing microglia sorted by flow cytometry. Aging microglia were characterized by the presence of lipofuscin granules, decreased processes complexity, altered granularity, and increased mRNA expression of both pro-inflammatory (TNFalpha, IL-1beta, IL-6) and anti-inflammatory (IL-10, TGFbeta1) cytokines. Following lipopolysaccharide (LPS) challenge (1 mg/kg, 3 h), aging microglia exhibit increased basal expression of TNFalpha, IL-1beta, IL-6, and IL-10. Yet, the fold-over-basal LPS response remained constant across age, implying that the inflammatory machinery in aging microglia is functional and adjusted to the basal state. Gender differences were not overall observed across the treatments (age, LPS). The low but sustained production of pro-inflammatory cytokines by aging microglia may have a profound impact in the brain aging process.
Asunto(s)
Envejecimiento/patología , Inflamación/patología , Microglía/patología , Animales , Células Cultivadas , Citocinas/biosíntesis , Femenino , Citometría de Flujo , Genes fms/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/ultraestructura , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Caracteres SexualesRESUMEN
E2a/Pbx1 is a fusion oncoprotein resulting from the t(1;19) translocation found in human pre-B acute lymphocytic leukemia and in a small number of acute T-lymphoid and myeloid leukemias. It was previously suggested that E2a/Pbx1 could cooperate with normal or oncogenic signaling pathways to immortalize myeloid and lymphoid progenitor cells. To address this question, we introduced the receptor of the macrophage-colony-stimulating factor (M-CSF-R) in pro-T cells immortalized by a conditional, estradiol-dependent, E2a/Pbx1-protein, and continuously proliferating in response to stem cell factor and interleukin-7. We asked whether M-CSF-R would be functional in an early T progenitor cell and influence the fate of E2a/Pbx1-immortalized cells. E2a-Pbx1 immortalized pro-T cells could proliferate and shifted from lymphoid to myeloid lineage after signaling through exogenously expressed M-CSF-R, irrespective of the presence of estradiol. However, terminal macrophage differentiation of the cells was obtained only when estradiol was withdrawn from cultures. This demonstrated that M-CSF-R is functional for proliferation and differentiation signaling in a T-lymphoid progenitor cell, which, in addition, unveiled myeloid potential of pro-T progenitors. Moreover, the block of differentiation induced by the E2a/Pbx1 oncogene could be modulated by hematopoietic cytokines such as M-CSF, suggesting plasticity of leukemic progenitor cells. Finally, additional experiments suggested that PU.1 and eight twenty-one transcriptional regulators might be implicated in the mechanisms of oncogenesis by E2a/Pbx1.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Transformación Celular Neoplásica , Proteínas de Homeodominio/fisiología , Proteínas de Fusión Oncogénica/fisiología , Linfocitos T/metabolismo , Animales , Western Blotting , Proliferación Celular , Estradiol/farmacología , Femenino , Citometría de Flujo , Genes fms/genética , Genes fms/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/genética , Interleucina-7/farmacología , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Células Mieloides/metabolismo , Proteínas de Fusión Oncogénica/genética , Fagocitosis , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factor de Células Madre/farmacología , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
Hematopoietic stem cells and multipotent progenitors exhibit low-level transcription and partial chromatin reorganization of myeloid cell-specific genes including the c-fms (csf1R) locus. Expression of the c-fms gene is dependent on the Ets family transcription factor PU.1 and is upregulated during myeloid differentiation, enabling committed macrophage precursors to respond to colony-stimulating factor 1. To analyze molecular mechanisms underlying the transcriptional priming and developmental upregulation of the c-fms gene, we have utilized myeloid progenitors lacking the transcription factor PU.1. PU.1 can bind to sites in both the c-fms promoter and the c-fms intronic regulatory element (FIRE enhancer). Unlike wild-type progenitors, the PU.1(-/-) cells are unable to express c-fms or initiate macrophage differentiation. When PU.1 was reexpressed in mutant progenitors, the chromatin structure of the c-fms promoter was rapidly reorganized. In contrast, assembly of transcription factors at FIRE, acquisition of active histone marks, and high levels of c-fms transcription occurred with significantly slower kinetics. We demonstrate that the reason for this differential activation was that PU.1 was required to promote induction and binding of a secondary transcription factor, Egr-2, which is important for FIRE enhancer activity. These data suggest that the c-fms promoter is maintained in a primed state by PU.1 in progenitor cells and that at FIRE PU.1 functions with another transcription factor to direct full activation of the c-fms locus in differentiated myeloid cells. The two-step mechanism of developmental gene activation that we describe here may be utilized to regulate gene activity in a variety of developmental pathways.
Asunto(s)
Ensamble y Desensamble de Cromatina , Regulación del Desarrollo de la Expresión Génica , Genes fms/genética , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Transcripción Genética/genética , Animales , Secuencia de Bases , Ensamble y Desensamble de Cromatina/genética , Desoxirribonucleasa I/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Elementos de Facilitación Genéticos , Histonas/metabolismo , Cinética , Metilación , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Células 3T3 NIH , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Proto-Oncogénicas/deficiencia , ARN Polimerasa II/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Transactivadores/deficiencia , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Colony stimulating factor-1 (CSF-1) is the primary regulator of the mononuclear phagocytic lineage acting through its transmembrane tyrosine kinase receptor, CSF-1R, that is the product of the c-fms proto-oncogene. Null mutations in either the ligand or the receptor genes result in a severe osteopetrosis as well as a number of other phenotypes, including reproductive defects and perturbations in organ development. The CSF-1R is also expressed in oocytes, myoblast progenitors, decidual, and trophoblastic cells. To distinguish cell type specific phenotypes, we have created a conditional allele of the Csf1r by placing LoxP sites around Exon 5 of the Csf1r gene in mice. Excision of this floxed sequence results in a null allele that in the homozygous state gives a phenotype indistinguishable of the complete Csf1r null mutant mouse. This conditional allele will prove extremely valuable to study the spatial and temporal roles of CSF-1R.
Asunto(s)
Genes fms/genética , Receptores del Factor Estimulante de Colonias/genética , Eliminación de Secuencia , Animales , Femenino , Marcación de Gen , Factor Estimulante de Colonias de Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , FenotipoRESUMEN
Myelodysplastic syndromes (MDS) are rare disorders in children. Molecular mechanisms underlying MDS in children are not yet completely understood. Considering the role of FMS and TP53 gene mutations in adult MDS patients, we analyzed mutations of these genes in a cohort of 35 children with MDS. Single-strand conformation polymorphism polymerase chain reaction analysis performed on FMS codon 969 and TP53 exons 5-9 showed no mutations in the analyzed sequences. Our results suggest that molecular mechanisms of MDS evolution in children are different from those in adults.
Asunto(s)
Genes fms/genética , Genes p53/genética , Mutación/genética , Síndromes Mielodisplásicos/genética , Preescolar , Codón/genética , Estudios de Cohortes , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Exones/genética , Humanos , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
MM-1 has been reported to repress the E-box-dependent transcription activity of c-Myc by recruiting histone deacetylase 1 complex via TIF1beta/KAP1. In this study, to identify target genes for c-Myc-MM-1-TIF1beta, we established rat-1 cells harboring the dominant-negative form of TIF1beta to abrogate the pathway from TIF1beta to MM-1-c-Myc. This cell line, in which transcription activity of c-Myc was activated, was found to be tumorigenic. By DNA-microarray analysis of this cell line, expression and promoter activity of the c-fms oncogene were found to be upregulated. Of the two promoters, pE1 and pE2, in the c-fms gene, pE1 promoter activity was found to be activated in an E-box-dependent manner.
Asunto(s)
Proteínas de Unión al ADN/farmacología , Fibroblastos/fisiología , Genes fms/genética , Genes myc/genética , Proteínas Proto-Oncogénicas c-myc/farmacología , Proteínas Represoras/farmacología , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Genes Reporteros , Luciferasas/genética , Datos de Secuencia Molecular , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Regiones Promotoras Genéticas/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteína 28 que Contiene Motivos TripartitoRESUMEN
BACKGROUND: Recent research found abnormal expression of the c-fms oncogene, which encodes the macrophage colony-stimulating factor receptor (CSF-1R), in several human carcinomas including hepatocellular carcinoma (HCC). But the relationship between the point mutation and abnormal expressing of c-fms oncogene in HCC was not clear. This study is to investigate the relationship between point mutation and abnormal expression of c-fms oncogene in hepatocellular carcinoma (HCC) and to clarify the mechanism of HCC. METHODS: The expression of c-fms oncogene at different levels of cell, protein and transcription was observed using immune histological ABC, Western blot and Northern blot. PCR-single strand conformation polymorphism and gene sequencing were used to detect the mutation of c-fms in HCC tissues and their surrounding tissues of 30 patients. RESULTS: The expression of c-fms was significantly higher in HCC tissues than in their surrounding tissues (P<0.01). Point mutation of Leu (TTG)-->Ser (TCG) at codon 301 of c-fms amino acids was observed in 21.4% (3/14) HCC tissues. No mutation of c-fms oncogene was detected in the surrounding cancerous tissues. CONCLUSION: Point mutation at codon 301 of c-fms oncogene is one of the mechanisms of abnormal over-expression in HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Genes fms/genética , Neoplasias Hepáticas/genética , Mutación Puntual , Adulto , Anciano , Secuencia de Bases , Northern Blotting , Western Blotting , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Femenino , Amplificación de Genes , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proto-Oncogenes/genética , ARN Mensajero/análisis , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
The mechanisms by which interleukin-6 (IL-6) family cytokines, which utilize the common receptor signaling subunit gp130, influence monocyte/macrophage development remain unclear. Here we have utilized macrophages devoid of either gp130-dependent STAT1/3 (gp130(Delta STAT/Delta STAT)) or extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinase (gp130(Y757F/Y757F)) activation to assess the individual contribution of each pathway to macrophage formation. While the inhibition by IL-6 of macrophage colony-stimulating factor (M-CSF)-induced colony formation observed in gp130(wt/wt) mice was abolished in gp130(Delta STAT/Delta STAT) mice, inhibition of macrophage colony formation was enhanced in gp130(Y757F/Y757F) mice. In gp130(Delta STAT/Delta STAT) bone marrow-derived macrophages (BMMs), both IL-6- and M-CSF-induced ERK1/2 tyrosine phosphorylation was enhanced. By contrast, tyrosine phosphorylation of ERK1/2 in response to M-CSF was reduced in gp130(Y757F/Y757F) BMMs, and the pattern of ERK1/2 activation in gp130 mutant BMMs correlated with their opposing responsiveness to M-CSF-induced proliferation. When compared to the level of expression in gp130(wt/wt) BMMs, c-fms expression was elevated in gp130(Delta STAT/Delta STAT) BMMs but reduced in gp130(Y757F/Y757F) BMMs. Finally, an ERK1/2 inhibitor suppressed M-CSF-induced BMM proliferation, and this result corresponded to a reduction in c-fms expression. Collectively, these results provide a functional and causal correlation between gp130-dependent ERK MAP kinase signaling and c-fms gene activation, a finding that provides a potential mechanism underlying the inhibition of M-CSF-dependent macrophage development by IL-6 family cytokines in mice.
Asunto(s)
Antígenos CD/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Genes fms/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Antígenos CD/genética , División Celular/efectos de los fármacos , Línea Celular , Receptor gp130 de Citocinas , Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Interleucina-6/farmacología , Péptidos y Proteínas de Señalización Intracelular , Macrófagos/citología , Glicoproteínas de Membrana/genética , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/metabolismo , Factor de Transcripción STAT3 , Transactivadores/metabolismo , Activación TranscripcionalRESUMEN
Colony stimulating factor (CSF-1) and its receptor (CSF-1R, product of c-fms proto-oncogene) were initially implicated as essential for normal monocyte development as well as for trophoblastic implantation. However, studies have demonstrated that CSF-1 and CSF-1R have additional roles in mammary gland development during pregnancy and lactation. This apparent role for CSF-1/CSF-1R in normal mammary gland development is very intriguing because this receptor/ligand pair has also been found to be important in the biology of breast cancer in which abnormal expression of CSF-1 and its receptor correlates with tumor cell invasiveness and adverse clinical prognosis. Recent findings also implicate tumor-produced CSF-1 in promotion of bone metastasis in breast cancer, and a certain membrane-associated form of CSF-1 appears to induce immunity against tumors. This review aims to summarize recent findings on the role of CSF-1 and its receptor in normal and neoplastic mammary development that may elucidate potential relationships of growth factor-induced biological changes in the breast during pregnancy and tumor progression.
Asunto(s)
Neoplasias de la Mama/metabolismo , Mama/fisiología , Factor Estimulante de Colonias de Macrófagos/fisiología , Animales , Mama/crecimiento & desarrollo , Mama/metabolismo , Femenino , Genes fms/genética , Humanos , Lactancia/metabolismo , Factor Estimulante de Colonias de Macrófagos/biosíntesis , Neoplasias Mamarias Experimentales/metabolismo , Embarazo , Proto-Oncogenes Mas , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Receptor de Factor Estimulante de Colonias de Macrófagos/fisiologíaAsunto(s)
Transformación Celular Neoplásica/genética , Células Eucariotas/metabolismo , Genes fms/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Animales , Ciclo Celular/genética , Humanos , Factor Estimulante de Colonias de Macrófagos/genética , Mitógenos/genética , Mitógenos/metabolismo , Proto-Oncogenes Mas , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/genéticaRESUMEN
The c-fms gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c-fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSs) were identified within mouse intron 2. Sequences of these DHSs were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fms intronic regulatory element (FIRE), which is even more highly conserved than the c-fms proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Sp1, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high- and low-level c-fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fms gene.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Genes fms/genética , Intrones/genética , Macrófagos/metabolismo , Regiones Promotoras Genéticas/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Células 3T3/metabolismo , Animales , Secuencia de Bases , Línea Celular , Desoxirribonucleasa I/metabolismo , Perfilación de la Expresión Génica , Genes Reporteros , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos Antisentido/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
To screen the receptor genes in renal cell carcinoma (RCC) associated with angiogenesis, we performed differential hybridization of the cDNA library of membrane-type protein tyrosine kinases (mPTKs). Three thousand plaques of a mPTKs-enriched cDNA library were screened with mPTKs mixture probes produced from hypervascular RCC tissues and RCC cell lines. Six different cDNA fragments of the PTK genes were isolated, and the sequence analysis showed that these represented cDNAs for TIE1, KDR, FMS, FGFR-4, JAK1 and HCK. Of these genes, the expression of TIE1, KDR, and FGFR-4 was studied in RCC tissue and cell lines by Northern blot analysis. We also investigated the expression of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their receptor FLT-1. In all the hypervascular RCC tissues, the amounts of mRNAs for KDR and FLT-1 were increased compared to adjacent normal tissues. The TIE1 and FGFR-4 genes were also overexpressed in most of the hypervascular RCC tissues, while no mRNA of KDR, FLT-1, or TIE1 could be detected in any of the four human RCC cell lines. The amounts of the VEGF and PlGF mRNAs were increased in hypervascular RCC tissues, while VEGF mRNA was detected in the four cell lines but PlGF mRNA was not. FGFR-4 mRNA was expressed in three of the four cell lines. These results suggest that KDR, FLT-1, PlGF and TIE1 mRNAs are present in the mesenchymal cells of RCC, while VEGF and FGFR-4 genes are expressed in RCC cells themselves in vivo.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/metabolismo , Neovascularización Patológica , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Superficie Celular/genética , Northern Blotting , Factores de Crecimiento Endotelial/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Biblioteca de Genes , Genes fms/genética , Humanos , Janus Quinasa 1 , Neoplasias Renales/patología , Linfocinas/genética , Factor de Crecimiento Placentario , Proteínas Gestacionales/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-hck , Receptor TIE-1 , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento/genética , Receptores TIE , Receptores de Factores de Crecimiento Endotelial Vascular , Células Tumorales Cultivadas , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
PU.1 is a unique regulatory protein required for the generation of both the innate and the adaptive immune system. It functions exclusively in a cell-intrinsic manner to control the development of granulocytes, macrophages, and B and T lymphocytes. We demonstrate that mutation of the PU.1 gene causes a severe reduction in myeloid (granulocyte/macrophage) progenitors. PU.1 -/- myeloid progenitors can proliferate in vitro in response to the multilineage cytokines interleukin-3 (IL-3), IL-6 and stem cell factor but are unresponsive to the myeloid-specific cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and M-CSF. The failure of PU.1 -/- progenitors to respond to G-CSF is bypassed by transient signaling with IL-3. In the presence of IL-3 and G-CSF, PU.1 -/- progenitors can differentiate into granulocytic precursors containing myeloperoxidase-positive granules. Thus PU.1 is not essential for specification of granulocytic precursors, but is required for their further differentiation. The failure of PU.1 -/- progenitors to respond to M-CSF is due to lack of c-fms gene transcription. Transduction of c-fms into PU.1 -/- myeloid progenitors bypasses the block to M-CSF-dependent proliferation but does not induce detectable macrophage differentiation. Therefore, PU. 1 appears to be essential for specification of monocytic precursors. Importantly, retroviral transduction of PU.1 into mutant progenitors restores responsiveness to myeloid-specific cytokines and development of mature granulocytes and macrophages. Thus PU.1 controls myelopoiesis by regulating both proliferation and differentiation pathways.
Asunto(s)
Citocinas/fisiología , Granulocitos/citología , Células Madre Hematopoyéticas/citología , Macrófagos/citología , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/fisiología , Animales , Diferenciación Celular/genética , División Celular/genética , Ensayo de Unidades Formadoras de Colonias , Femenino , Regulación de la Expresión Génica , Genes fms/genética , Factor Estimulante de Colonias de Granulocitos/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Granulocitos/efectos de los fármacos , Granulocitos/fisiología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Interleucina-3/farmacología , Interleucina-6/farmacología , Factor Estimulante de Colonias de Macrófagos/fisiología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Masculino , Ratones , Ratones Noqueados , Mutación , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Retroviridae/genética , Factor de Células Madre/farmacología , Transactivadores/deficiencia , Transactivadores/genética , Transducción GenéticaRESUMEN
The FMS proto-oncogene encodes the cell surface receptor for colony stimulating factor-1 (CSF-1). Mutations of the FMS gene at codon 969, in the C-terminal region of the gene, have been detected in haematological malignancies. To ascertain the biological significance of a mutation at this codon, we have used a murine haematopoietic cell line, FDC-P1, containing a mutation at codon 969 that results in a phenylalanine replacing a tyrosine. FMS 969 mutant cells and v-fms transfected cells conferred interleukin 3 (IL-3) independent stimulation of FDC-P1 cells, whereas cells transfected with a wild-type FMS construct required exogenous IL-3 for growth. FDC-P1 cells containing a FMS 969 mutation and v-fms transfected cells were tumorigenic in nude mice. Binding studies with radioidonated CSF-1 revealed saturable specific binding in FMS wild-type cells with a Km of 0.9 mM; however, mutant FMS-containing cells did not display saturation kinetics, but instead exhibited a linear relationship between ligand concentration and amount bound. Constitutive expression of FOS was detected in 969 mutant cells in the absence of exogenous CSF-1, a phenotype that was only inducible in wild-type cells in response to CSF-1. FOS and JUNB expression by v-FMS transfected cells showed a similar pattern to FMS wild-type cells. This mutation has been detected in patients with haematological malignancies, and illustrates that the pathway of FMS 969 phenylalanine mutations and v-fms induced pathogenesis can be distinguished. These data indicate that there is a biological role for FMS codon 969 phenylalanine mutation which results in transformation of FDC-P1 cells.
Asunto(s)
Codón/genética , Genes fms/genética , Mutación Puntual/genética , Mutación Puntual/fisiología , Animales , Northern Blotting , Pruebas de Carcinogenicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Genes fos/efectos de los fármacos , Genes fos/genética , Genes jun/efectos de los fármacos , Genes jun/genética , Células Madre Hematopoyéticas/fisiología , Interleucina-3/farmacología , Radioisótopos de Yodo/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Ratones Desnudos , ARN/análisis , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesisRESUMEN
Macrophage colony-stimulating factor (M-CSF) is a hematopoietin whose actions are essential for growth and survival of macrophages, placental development, ramification of microglia and tumor progression. The expression of the receptor for macrophage colony-stimulating factor (c-fms) is regulated by two distinct promoters: distal and proximal. The distal promoter is active in trophoblasts during embryogenesis and the proximal promoter directs expression to the cells of myeloid lineage. Here we report the generation of transgenic mice expressing beta-galactosidase under the control of the human proximal c-fms promoter and demonstrate the promoter activity in astrocytes, cells of neurological origin that partially take over the role of the macrophages in the central nervous system. Enzymatic activity of beta-galactosidase was detected in homogenated spleen, bone marrow and brain and in the cell extracts from peritoneal macrophages of transgenic mice. Immunohistochemical staining of brain showed the presence of beta-galactosidase in astrocytes. We hypothesize that M-CSF released by astrocytes, upon stimulation by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) or interleukin-1 (IL-1), regulates the expression of its own receptor.
Asunto(s)
Astrocitos/fisiología , Factor Estimulante de Colonias de Macrófagos/farmacología , Regiones Promotoras Genéticas/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/fisiología , Animales , Astrocitos/metabolismo , Genes fms/genética , Humanos , Ratones , Ratones TransgénicosRESUMEN
Vascular medial smooth muscle cells migrate, proliferate and transform to foam cells in the process of atherosclerosis. We have reported that the intimal smooth muscle cells express proto-oncogene c-fms, a characteristic gene of monocyte-macrophages, which is not normally expressed in medial smooth muscle cells. In the present study, we demonstrated that combinations of platelet-derived growth factor (PDGF)-BB and either epidermal growth factor (EGF) or fibroblast growth factor (FGF) induced high expression of c-fms in normal human medial smooth muscle cells to the level of intimal smooth muscle cells or monocyte-derived macrophages, whereas c-fms expression by PDGF-BB alone was 1/10 and both EGF and FGF had no independent effect on c-fms expression. By contrast, interferon (IFN)-gamma and macrophage colony-stimulating factor (M-CSF) suppressed the induction of c-fms expression. These results indicate that multiple growth factors and cytokines may play a role in the phenotypic transformation of medial smooth muscle cells to intimal smooth muscle cells in atherosclerotic lesions by altering c-fms expression.
Asunto(s)
Citocinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes fms/genética , Proteínas de la Membrana , Músculo Liso Vascular/fisiología , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Receptores de Lipoproteína , Aorta/citología , Arteriosclerosis/etiología , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Interferón gamma/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Desarrollo de Músculos , Músculo Liso Vascular/crecimiento & desarrollo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proto-Oncogenes Mas , ARN Mensajero/análisis , Receptores Inmunológicos/análisis , Receptores Depuradores , Receptores Depuradores de Clase B , Túnica Íntima/citología , Túnica Íntima/crecimiento & desarrollo , Túnica Media/citología , Túnica Media/crecimiento & desarrolloRESUMEN
In order to better understand the role of transcription in cellular processing of damage in specific DNA sequences, we have used an in vitro differentiation system to modulate the activity of the MYC gene. When human HL60 promyelocytic cells differentiate in vitro, the transcriptional activity of the MYC gene is down-regulated. We have shown that in the expressed MYC gene, 56% of UV-induced cyclobutane pyrimidine dimers (CPDs) are removed within 18 h and the transcribed strand is selectively repaired. However, late in differentiation, when the MYC gene is maximally down-regulated, only 15% of the CPDs are removed within the same period. During early differentiation, the MYC gene is regulated by a block to transcription elongation at the 5' end of the first intron. Our results reveal no significant difference in the rate of CPD removal between the restriction fragments upstream and downstream of this elongation block. Furthermore, both strands of each fragment exhibit similar repair characteristics. In contrast, the constitutively expressed FMS gene exhibits proficient removal of CPD in both the differentiated and undifferentiated cells. Furthermore, the repair appears to be more proficient at the 5' end (exon 1) than in the 3' end of the gene about 35 kilobases downstream from exon 1. Since efficient repair of the active FMS gene is maintained in the differentiated cells the loss of repair competence seen in MYC is more likely associated with its reduced transcriptional activity than with a decrease in the overall repair capacity of the terminally differentiated cells.