RESUMO
Cytokines stimulate granulopoiesis through signaling via receptors whose expression is controlled by lineage-specific transcription factors. Previously, we demonstrated that granulocyte colony-stimulating factor (G-CSF) receptor mRNA was undetectable and granulocyte maturation blocked in CCAAT enhancer binding protein alpha (C/EBPalpha)-deficient mice. This phenotype is distinct from that of G-CSF receptor-/- mice, suggesting that other genes are likely to be adversely affected by loss of C/EBPalpha. Here we demonstrate loss of interleukin 6 (IL-6) receptor and IL-6-responsive colony-forming units (CFU-IL6) in C/EBPalpha-/- mice. The observed failure of granulopoiesis could be rescued by the addition of soluble IL-6 receptor and IL-6 or by retroviral transduction of G-CSF receptors, demonstrating that loss of both of these receptors contributes to the absolute block in granulocyte maturation observed in C/EBPalpha-deficient hematopoietic cells. The results of these and other studies suggest that additional C/EBPalpha target genes, possibly other cytokine receptors, are also important for the block in granulocyte differentiation observed in vivo in C/EBPalpha-deficient mice.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Granulócitos/fisiologia , Hematopoese , Proteínas Nucleares/fisiologia , Receptores de Fator Estimulador de Colônias de Granulócitos/biossíntese , Receptores de Interleucina-6/biossíntese , Fatores de Transcrição/fisiologia , Regulação para Cima/fisiologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular/genética , Ensaio de Unidades Formadoras de Colônias , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Elementos Facilitadores Genéticos , Feto , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interleucina-6/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/fisiologia , Camundongos , Camundongos Knockout , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Receptores de Fator Estimulador de Colônias de Granulócitos/antagonistas & inibidores , Receptores de Fator Estimulador de Colônias de Granulócitos/deficiência , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/deficiência , Receptores de Interleucina-6/genética , Solubilidade , Fatores de Transcrição/genética , Regulação para Cima/genéticaRESUMO
Recent work has demonstrated that p56lck, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59fyn, p55fgr, and p56hck, but not into p60src. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59fyn and p56lck with serine indicated that Cys3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol-anchored proteins. Introduction of Cys3 into p60src led to its palmitoylation. p59fyn but not p60src partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence Met-Gly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.
Assuntos
Caveolinas , Membrana Celular/metabolismo , Cisteína/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Acilação , Sequência de Aminoácidos , Antígenos CD/análise , Antígenos CD/metabolismo , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Antígenos CD55 , Caveolina 1 , Membrana Celular/química , Cisteína/química , Cisteína/metabolismo , Células HeLa , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/análise , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Miristatos/metabolismo , Palmitatos/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-fyn , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismoRESUMO
The role of mutations of the granulocyte colony-stimulating factor receptor (G-CSFR) in the pathogenesis of severe congenital neutropenia (SCN) and the subsequent development of acute myeloid leukemia (AML) is controversial. Mice carrying a targeted mutation of their G-CSFR that reproduces the mutation found in a patient with SCN and AML have been generated. The mutant G-CSFR allele is expressed in a myeloid-specific fashion at levels comparable to the wild-type allele. Mice heterozygous or homozygous for this mutation have normal levels of circulating neutrophils and no evidence for a block in myeloid maturation, indicating that resting granulopoiesis is normal. However, in response to G-CSF treatment, these mice demonstrate a significantly greater fold increase in the level of circulating neutrophils. This effect appears to be due to increased neutrophil production as the absolute number of G-CSF-responsive progenitors in the bone marrow and their proliferation in response to G-CSF is increased. Furthermore, the in vitro survival and G-CSF-dependent suppression of apoptosis of mutant neutrophils are normal. Despite this evidence for a hyperproliferative response to G-CSF, no cases of AML have been detected to date. These data demonstrate that the G-CSFR mutation found in patients with SCN is not sufficient to induce an SCN phenotype or AML in mice.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Hematopoese/efeitos dos fármacos , Neutropenia/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/deficiência , Doença Aguda , Animais , Apoptose/efeitos dos fármacos , Medula Óssea/patologia , Divisão Celular/efeitos dos fármacos , Suscetibilidade a Doenças , Feminino , Genótipo , Humanos , Leucemia Mieloide/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndromes Mielodisplásicas/etiologia , Neutropenia/congênito , Neutropenia/patologia , Neutrófilos/patologia , Receptores de Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Receptores de Fator Estimulador de Colônias de Granulócitos/genéticaRESUMO
Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is widely used to treat neutropenia. In addition to stimulating polymorphonuclear neutrophil (PMN) production, G-CSF may have significant effects on PMN function. Because G-CSF receptor (G-CSFR)-deficient mice do not have the expected neutrophilia after administration of human interleukin-8 (IL-8), we examined the effect of the loss of G-CSFR on IL-8-stimulated PMN function. Compared with wild-type PMNs, PMNs isolated from G-CSFR-deficient mice demonstrated markedly decreased chemotaxis to IL-8. PMN emigration into the skin of G-CSFR-deficient mice in response to IL-8 was also impaired. Significant chemotaxis defects were also seen in response to N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, or macrophage inflammatory protein-2. The defective chemotactic response to IL-8 does not appear to be due to impaired chemoattractant receptor function, as the number of IL-8 receptors and chemoattractant-induced calcium influx, actin polymerization, and release of gelatinase B were comparable to those of wild-type PMNs. Chemoattractant-induced adhesion of G-CSFR-deficient PMNs was significantly impaired, suggesting a defect in beta2-integrin activation. Collectively, these data demonstrate that selective defects in PMN activation are present in G-CSFR-deficient mice and indicate that G-CSF plays an important role in regulating PMN chemokine responsiveness.
Assuntos
Fatores Quimiotáticos/farmacologia , Ativação de Neutrófilo , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Actinas/metabolismo , Animais , Antígenos CD/análise , Cálcio/metabolismo , Adesão Celular/genética , Degranulação Celular , Quimiocina CXCL2 , Quimiocinas/farmacologia , Quimiotaxia de Leucócito , Colagenases/metabolismo , Interleucina-8/farmacologia , Metaloproteinase 9 da Matriz , Camundongos , Camundongos Mutantes , Monocinas/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Interleucina/análise , Receptores de Interleucina-8A , Pele/imunologia , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The fgr proto-oncogene encodes a nonreceptor protein-tyrosine kinase, designated p55c-fgr. In this study, we have isolated human fgr cDNA molecules from normal monocyte mRNA templates. Nucleotide sequence analysis of the longest fgr cDNA revealed a 5' untranslated region of 927 bp which included two Alu-like repeats as well as three translation stop codons immediately upstream of the initiator for p55c-fgr synthesis. Within genomic DNA, these sequences were distributed over 13 kbp as three distinct 5' untranslated exons. Previous studies have shown that Epstein-Barr virus (EBV) increases c-fgr mRNA levels in B lymphocytes. By comparing the nucleotide sequence reported for transcripts isolated from EBV-infected B lymphocytes with those of our monocyte cDNA as well as genomic DNA, we identified a novel untranslated exon utilized only in EBV-infected cells. The transcriptional initiation sites of fgr mRNA expressed in EBV-converted cells were mapped and shown to reside within a region identified as an intron for fgr mRNA that is expressed in normal myelomonocytic cells. Furthermore, the region of the fgr locus upstream of the novel exon displayed properties of a transcriptional promoter when transfected into heterologous cells. We conclude from all of these findings that activation of the fgr gene by EBV is achieved by mechanisms distinct from those normally regulating its programmed expression in myelomonocytic cells.
Assuntos
Linfócitos B/fisiologia , Monócitos/fisiologia , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Sequência de Bases , Transformação Celular Viral , Clonagem Molecular , DNA/genética , Éxons , Genes , Herpesvirus Humano 4 , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proto-Oncogene Mas , RNA Mensageiro/genética , Mapeamento por Restrição , Quinases da Família srcRESUMO
Traditional response criteria in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are based on bone marrow morphology and may not accurately reflect clonal tumor burden in patients treated with non-cytotoxic chemotherapy. We used next-generation sequencing of serial bone marrow samples to monitor MDS and AML tumor burden during treatment with epigenetic therapy (decitabine and panobinostat). Serial bone marrow samples (and skin as a source of normal DNA) from 25 MDS and AML patients were sequenced (exome or 285 gene panel). We observed that responders, including those in complete remission (CR), can have persistent measurable tumor burden (that is, mutations) for at least 1 year without disease progression. Using an ultrasensitive sequencing approach, we detected extremely rare mutations (equivalent to 1 heterozygous mutant cell in 2000 non-mutant cells) months to years before their expansion at disease relapse. While patients can live with persistent clonal hematopoiesis in a CR or stable disease, ultimately we find evidence that expansion of a rare subclone occurs at relapse or progression. Here we demonstrate that sequencing of serial samples provides an alternative measure of tumor burden in MDS or AML patients and augments traditional response criteria that rely on bone marrow blast percentage.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Evolução Clonal/genética , Epigênese Genética/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/patologia , Exoma , Feminino , Genes p53 , Sequenciamento de Nucleotídeos em Larga Escala , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Mutação , Síndromes Mielodisplásicas/diagnóstico , Polimorfismo de Nucleotídeo Único , Indução de Remissão , Resultado do Tratamento , Carga TumoralRESUMO
In this study, we have characterized the 5' region of the human c-fgr proto-oncogene and identified the major myelomonocytic c-fgr promoter. Seven distinct 5' untranslated exons were identified and localized to a region extending 13 kb upstream from the first coding exon. Two major promoters were identified, one utilized exclusively in Epstein-Barr virus (EBV)-infected B-lymphocyte cell lines, and the other functional only in myelomonocytic cells. Differential promoter utilization and alternative splicing of the 5' untranslated exons give rise to at least six distinct c-fgr mRNA species that differ only in their 5' untranslated regions. Two major mRNAs were identified, c-fgr A and c-fgr 4; these two mRNAs were detected exclusively in EBV-infected B-lymphocyte cell lines and myelomonocytic cells respectively. We have previously demonstrated that c-fgr is transcriptionally activated in U937 cells treated with either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or cycloheximide (CHX). We now show that a DNA fragment extending from -772 to +97 (with respect to the transcription initiation site upstream from exon M4) is responsive to TPA but not CHX treatment in U937 cells. These results suggest that TPA and CHX induce c-fgr mRNA accumulation by different mechanisms.
Assuntos
Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas/genética , Sequência de Bases , Mapeamento Cromossômico , Cicloeximida/farmacologia , Éxons/genética , Humanos , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Proto-Oncogene Mas , RNA/análise , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas , Quinases da Família srcRESUMO
1,25-dihydroxyvitamin D3 [1,25(OH)2D] is known to stimulate maturation of the human promyelocytic line HL-60 and murine bone marrow precursor cells along a monocyte/macrophage pathway. In this study, the steroid's effects on the PLB-985 leukemic line were examined. We found that 1,25(OH)2D induces monocytic differentiation of PLB-985 as manifested by morphological appearance, histochemical staining, and changes in cell surface antigen expression. Additionally, there was acquisition of functional monocyte characteristics including phagocytic activity and superoxide anion production via the respiratory burst pathway. Steady state levels of mRNA derived from the leukocyte-specific proto-oncogene c-fgr were also increased by the steroid. Thus, 1,25(OH2)D effectively differentiates PLB-985 cells along a monocytic pathway, providing a useful model of macrophage differentiation.
Assuntos
Calcitriol/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Monócitos/patologia , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Leucemia Mieloide/metabolismo , Monócitos/metabolismo , Fagocitose/efeitos dos fármacos , Proto-Oncogene Mas , RNA Mensageiro/metabolismo , Superóxidos/metabolismo , Células Tumorais Cultivadas , Quinases da Família srcRESUMO
Hematopoietic progenitor cells (HPC) can be mobilized from the bone marrow into the peripheral circulation in response to diverse stimuli, including hematopoietic growth factors, cytotoxic agents, and certain chemokines. Despite significant differences in their biologic activities, these stimuli result in the mobilization of HPC with a similar phenotype, suggesting that a common mechanism for mobilization may exist. To explore the mechanisms of granulocyte colony-stimulating factor (G-CSF)-induced mobilization, we examined HPC mobilization in mice that are genetically deficient for the G-CSF receptor (G-CSFR). Their response was determined to each of three major types of mobilizing stimuli: cytotoxic agents (cyclophosphamide), chemokines (Interleukin-8[IL-8]), and hematopoietic growth factors (G-CSF, fit-3 ligand, and IL-12). These studies demonstrate that the G-CSFR is required for mobilization in response to cyclophosphamide and IL-8, but not fit-3 ligand or IL-12, and suggest that the G-CSFR may play an important and previously unexpected role in HPC migration.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Animais , Células da Medula Óssea , Citocinas/farmacologia , Humanos , Proteínas de Membrana/farmacologia , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologiaRESUMO
Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte colony-stimulating factor (G-CSF) is often induced with infection and has a key role in the stress granulopoiesis response. However, its effects on HSCs are less clear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect associated with induction of Toll-like receptor (TLR) expression and signaling. The G-CSF-mediated expansion of HSCs is reduced in mice lacking TLR2, TLR4 or the TLR signaling adaptor MyD88. Induction of HSC quiescence is abrogated in mice lacking MyD88 or in mice treated with antibiotics to suppress intestinal flora. Finally, loss of TLR4 or germ-free conditions mitigates the G-CSF-mediated HSC repopulating defect. These data suggest that low-level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Transdução de Sinais/fisiologia , Receptores Toll-Like/fisiologia , Animais , Células-Tronco Hematopoéticas/fisiologia , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/fisiologia , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/fisiologiaRESUMO
Recent studies suggest that most cases of myelodysplastic syndrome (MDS) are clonally heterogeneous, with a founding clone and multiple subclones. It is not known whether specific gene mutations typically occur in founding clones or subclones. We screened a panel of 94 candidate genes in a cohort of 157 patients with MDS or secondary acute myeloid leukemia (sAML). This included 150 cases with samples obtained at MDS diagnosis and 15 cases with samples obtained at sAML transformation (8 were also analyzed at the MDS stage). We performed whole-genome sequencing (WGS) to define the clonal architecture in eight sAML genomes and identified the range of variant allele frequencies (VAFs) for founding clone mutations. At least one mutation or cytogenetic abnormality was detected in 83% of the 150 MDS patients and 17 genes were significantly mutated (false discovery rate ≤0.05). Individual genes and patient samples displayed a wide range of VAFs for recurrently mutated genes, indicating that no single gene is exclusively mutated in the founding clone. The VAFs of recurrently mutated genes did not fully recapitulate the clonal architecture defined by WGS, suggesting that comprehensive sequencing may be required to accurately assess the clonal status of recurrently mutated genes in MDS.
Assuntos
Mutação , Síndromes Mielodisplásicas/genética , Feminino , Frequência do Gene , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , RecidivaRESUMO
Under normal conditions, the great majority of hematopoietic stem/progenitors cells (HSPCs) reside in the bone marrow. The number of HSPCs in the circulation can be markedly increased in response to a number of stimuli, including hematopoietic growth factors, myeloablative agents and environmental stresses such as infection. The ability to 'mobilize' HSPCs from the bone marrow to the blood has been exploited clinically to obtain HSPCs for stem cell transplantation and, more recently, to stimulate therapeutic angiogenesis at sites of tissue ischemia. Moreover, there is recent interest in the use of mobilizing agents to sensitize leukemia and other hematopoietic malignancies to cytotoxic agents. Key to optimizing clinical mobilizing regimens is an understanding of the fundamental mechanisms of HSPC mobilization. In this review, we discuss recent advances in our understanding of the mechanisms by which granulocyte colony-stimulating factor (G-CSF), the prototypical mobilizing agent, induces HSPC mobilization.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Neoplasias Hematológicas/terapia , HumanosRESUMO
The role of cytokines in the control of hematopoietic cell differentiation remains controversial. Two general models for the cytokine control of hematopoietic differentiation have been proposed. In the stochastic model, cytokines provide proliferative and survival signals to the differentiating hematopoietic cell, but they do not provide specific lineage commitment signals. In the instructive model, cytokines transmit specific signals to multipotent hematopoietic cells, thereby directing lineage commitment. To distinguish between these two models with respect to granulocyte colony-stimulating factor (G-CSF) and granulocytic differentiation, we used the 32Dcl3 cell line, which is capable of differentiating into granulocytes in response to G-CSF, 32D cells transfected with either bcl-2 or bcl-XL showed prolonged survival in medium containing no cytokine supplement. Cells surviving in these cultures developed the segmented nuclei characteristic of mature neutrophils. However, no induction of myeloperoxidase activity or increase in cathepsin G transcripts were detected. These data support a hybrid model for the role of G-CSF in granulocytic differentiation; although some features of granulocytic differentiation, namely nuclear segmentation, do not require G-CSF and appear therefore to be preprogrammed in 32D cells, the complete maturation of these cells to granulocytes appears to be dependent on G-CSF.
Assuntos
Apoptose , Fator Estimulador de Colônias de Granulócitos/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Grânulos Citoplasmáticos/ultraestrutura , Genes Reporter , Fator Estimulador de Colônias de Granulócitos/farmacologia , Granulócitos/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Recombinantes de Fusão/biossíntese , Transfecção , Proteína bcl-XRESUMO
The proto-oncogene c-fgr is a member of the c-src gene family of cytoplasmic tyrosine kinases. Previous studies have suggested that it is normally expressed in neutrophils, monocytes, macrophages, and natural killer cells. c-fgr is also expressed in the B cells of certain lymphoproliferative disorders, namely, Epstein-Barr virus-associated lymphoproliferative disease, and in chronic lymphocytic leukemia, but it has not previously been detected in normal or reactive human lymphoid tissue. In this study we have determined the pattern of p55c-fgr protein expression in normal human hematopoietic and lymphoid tissues at the single-cell level using immunohistochemical and immunofluorescent techniques. We show that p55c-fgr expression is developmentally regulated with high-level expression first evident at the myelocyte stage of myeloid differentiation. In addition, we show that p55c-fgr is expressed in circulating B lymphocytes isolated from chronic lymphocytic leukemia patients but is not expressed in normal circulating B lymphocytes. Surprisingly, p55c-fgr is also expressed in a subpopulation of normal B lymphocytes, the mantle zone B lymphocytes. This demonstration that p55c-fgr is expressed in a normal B-lymphocyte subpopulation suggests that its expression in certain B-cell lymphoproliferative disorders may be an indirect consequence of, rather than a primary cause of, the neoplastic transformation process.
Assuntos
Subpopulações de Linfócitos B/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Linfonodos/citologia , Tecido Linfoide/citologia , Proteínas Proto-Oncogênicas/biossíntese , Proto-Oncogenes , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Eritroblástica Aguda/patologia , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-hck , Células Tumorais Cultivadas , Quinases da Família srcRESUMO
Hematopoietic progenitor cells (HPC) can be mobilized from the bone marrow into the peripheral circulation in response to a number of stimuli including hematopoietic growth factors, cytotoxic agents, and certain chemokines. Despite significant differences in their biological activities, these stimuli result in the mobilization of HPC with a similar phenotype, suggesting that a common mechanism for mobilization may exist. In this study, the role of granulocyte colony-stimulating factor (G-CSF) in progenitor mobilization was examined using G-CSF receptor (G-CSFR)-deficient mice. In contrast to wild-type mice, no increase in circulating colony-forming cells (CFU-C), CD34+ lineage- progenitors, or day 12 colony-forming unit-spleen progenitors (CFU-S) was detected in G-CSFR-deficient mice after cyclophosphamide administration. This defect was not due to a failure to regenerate HPC following cyclophosphamide administration as the number of CFU-C in the bone marrow of G-CSFR-deficient mice was increased relative to wild-type mice. Likewise, no increase in circulating CFU-C was detected in G-CSFR-deficient mice following interleukin-8 (IL-8) administration. In contrast, mobilization of HPC in response to flt-3 ligand was nearly normal. These results show that the G-CSFR is required for mobilization in response to cyclophosphamide or IL-8 but not flt-3 ligand and suggest that the G-CSFR may play an important and previously unexpected role in HPC migration.
Assuntos
Ciclofosfamida/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Interleucina-8/farmacologia , Proteínas de Membrana/farmacologia , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Animais , Contagem de Células Sanguíneas , Células da Medula Óssea/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Fator Estimulador de Colônias de Granulócitos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Fator Estimulador de Colônias de Granulócitos/deficiência , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Proteínas Recombinantes/farmacologia , Baço/citologiaRESUMO
The mechanisms that regulate hematopoietic progenitor cell (HPC) mobilization from the bone marrow to blood have not yet been defined. HPC mobilization by granulocyte colony-stimulating factor (G-CSF), cyclophosphamide (CY), or interleukin-8 but not flt-3 ligand is markedly impaired in G-CSF receptor-deficient (G-CSFR-deficient) mice. G-CSFR is expressed on mature hematopoietic cells, HPCs, and stromal cells, which suggests that G-CSFR signals in one or more of these cell types was required for mobilization by these agents. To define the cell type(s) responsible for G-CSF-dependent mobilization, a series of chimeric mice were generated using bone marrow transplantation. Mobilization studies in these chimeras demonstrated that expression of the G-CSFR on transplantable hematopoietic cells but not stromal cells is required for CY- or G-CSF-induced mobilization. Moreover, in irradiated mice reconstituted with both wild type and G-CSFR-deficient bone marrow cells, treatment with CY or G-CSF resulted in the equal mobilization of both types of HPCs. This result held true for a broad spectrum of HPCs including colony-forming cells, CD34(+) lineage(-) and Sca(+) lineage(-) cells, and long-term culture initiating cells. Collectively, these data provide the first definitive evidence that expression of the G-CSFR on HPCs is not required for their mobilization by G-CSF and suggest a model in which G-CSFR-dependent signals act in trans to mobilize HPCs from the bone marrow.
Assuntos
Transplante de Medula Óssea , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Animais , Fator Estimulador de Colônias de Granulócitos/fisiologia , Células-Tronco Hematopoéticas/patologia , Camundongos , Quimeras de TransplanteRESUMO
Multiple hematopoietic cytokines can stimulate granulopoiesis; however, their relative importance in vivo and mechanisms of action remain unclear. We recently reported that granulocyte colony-stimulating factor receptor (G-CSFR)-deficient mice have a severe quantitative defect in granulopoiesis despite which phenotypically normal neutrophils were still detected. These results confirmed a role for the G-CSFR as a major regulator of granulopoiesis in vivo, but also indicated that G-CSFR independent mechanisms of granulopoiesis must exist. To explore the role of interleukin-6 (IL-6) in granulopoiesis, we generated IL-6 x G-CSFR doubly deficient mice. The additional loss of IL-6 significantly worsened the neutropenia present in young adult G-CSFR-deficient mice; moreover, exogenous IL-6 stimulated granulopoiesis in vivo in the absence of G-CSFR signals. Near normal numbers of myeloid progenitors were detected in the bone marrow of IL-6 x G-CSFR-deficient mice and their ability to terminally differentiate into mature neutrophils was observed. These results indicate that IL-6 is an independent regulator of granulopoiesis in vivo and show that neither G-CSFR or IL-6 signals are required for the commitment of multipotential progenitors to the myeloid lineage or for their terminal differentiation.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Fator Estimulador de Colônias de Granulócitos/fisiologia , Hematopoese/fisiologia , Interleucina-6/fisiologia , Neutropenia/fisiopatologia , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Receptores de Interleucina-6/fisiologia , Animais , Feminino , Células-Tronco Hematopoéticas/citologia , Interleucina-6/sangue , Interleucina-6/deficiência , Interleucina-6/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutropenia/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/deficiência , Receptores de Fator Estimulador de Colônias de Granulócitos/genéticaRESUMO
We have generated mice carrying a homozygous null mutation in the granulocyte colony-stimulating factor receptor (G-CSFR) gene. G-CSFR-deficient mice have decreased numbers of phenotypically normal circulating neutrophils. Hematopoietic progenitors are decreased in the bone marrow, and the expansion and terminal differentiation of these progenitors into granulocytes is impaired. Neutrophils isolated from G-CSFR-deficient mice have an increased susceptibility to apoptosis, suggesting that the G-CSFR may also regulate neutrophil survival. These data confirm a role for the G-CSFR as a major regulator of granulopoiesis in vivo and provide evidence that the G-CSFR may regulate granulopoiesis by several mechanisms. However, the data also suggest that G-CSFR-independent mechanisms of granulopoiesis must exist.