RESUMO
The presence of replication-competent retrovirus (RCR) in retroviral-based gene therapy products poses a potential safety risk for patients. Therefore, RCR testing of clinical gene therapy products and monitoring of patients enrolled in gene therapy trials is required to assure viral safety. The requirement to test ex vivo-transduced cells originates from the presumed amplification of adventitious RCR during the transduction procedure. However, data on the capacity of different cell types to do so are lacking. In this study, we sought to analyze the amplification potential of primary human T lymphocytes after infection with amphotropic MLV-based RCR. The total number of viral particles produced after 1 or 2 weeks was measured by a quantitative 4070A env-specific RT-PCR assay. The fraction of infectious replication-competent viral particles was analyzed in the PG-4 S+L- assay. From this study, we conclude that the total number of viral particles RCR produced by T lymphocytes is 2-4 logs lower than the number produced by NIH-3T3 cells. Surprisingly, less than 1% of the viral particles produced by primary T lymphocytes appeared to be infectious, while nearly all virions produced by NIH-3T3 were. We conclude that primary human T lymphocytes are low producers of MLV-based amphotropic RCR.
Assuntos
Terapia Genética/efeitos adversos , Vírus da Leucemia Murina de Moloney/fisiologia , Linfócitos T/virologia , Replicação Viral , Expressão Gênica , Vetores Genéticos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução Genética/métodosRESUMO
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).
Assuntos
Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Transativadores/metabolismo , Proteínas Supressoras de Tumor , Animais , Sítios de Ligação , Diferenciação Celular , Divisão Celular , Sobrevivência Celular , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas de Ligação a DNA/genética , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/genética , Células HeLa , Humanos , Ligantes , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Mutagênese , Neutrófilos/citologia , Regiões Promotoras Genéticas , Fator de Transcrição STAT3 , Células-Tronco/citologia , Transativadores/genética , Transfecção , Células Tumorais CultivadasRESUMO
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.
Assuntos
Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Neutropenia/congênito , Neutropenia/tratamento farmacológico , Mutação Puntual , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Células da Medula Óssea/metabolismo , Divisão Celular , Relação Dose-Resposta a Droga , Feminino , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Lactente , Ligantes , Modelos Moleculares , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transdução de SinaisRESUMO
Acquired mutations truncating the C-terminal domain of the granulocyte colony-stimulating factor receptor (G-CSF-R) are found in about 20% of severe congenital neutropenia (SCN) patients, with this cohort of patients predisposed to acute myeloid leukemia (AML). In myeloid cells, such mutations act in a dominant-negative manner leading to hyperproliferation and lack of differentiation in response to G-CSF. However, why these truncated receptors are dominant in function over wild-type receptors has remained unclear. We report that ligand-induced internalization of truncated G-CSF-R is severely impaired compared with the wild-type receptor, which results in sustained activation of STAT proteins. Strikingly, in cells coexpressing both truncated and wild-type forms, the truncated receptors acted dominantly with regard to both internalization and sustained activation. Site-directed mutagenesis of the C-terminus showed that receptor tyrosines in this region were dispensable for internalization, whereas a di-leucine-containing motif in Box B3 played some role. However, loss of the di-leucine motif was not the critical determinant of the sustained activation status of truncated receptors. These data suggest that defective internalization, leading to extended receptor activation, is a major cause of the dominant hyperproliferative effect of truncated G-CSF receptors, which is only partially due to the loss of a di-leucine motif present in the Box B3 region of the full-length receptor.
Assuntos
Leucemia Mieloide Aguda/genética , Proteínas do Leite , Mutação , Neutropenia/congênito , Neutropenia/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Animais , Citocalasina D/farmacologia , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Leucina , Camundongos , Mutagênese Sítio-Dirigida , Receptores de Fator Estimulador de Colônias de Granulócitos/química , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Fator de Transcrição STAT5 , Relação Estrutura-Atividade , Transativadores/metabolismo , Células Tumorais Cultivadas , TirosinaRESUMO
The granulocyte colony-stimulating factor receptor (G-CSF-R) activates multiple STAT proteins. Although the membrane-proximal cytoplasmic region of the G-CSF-R is necessary and sufficient for activation of STAT1 and STAT5, activation of STAT3 requires the membrane distal region that contains four tyrosines. Although one of these (Y704) has previously been shown to be involved in STAT3 activation from a truncated G-CSF-R derived from a patient with severe chronic neutropenia (SCN), this tyrosine is not required for STAT3 activation by the full-length G-CSF-R. To investigate possible alternative mechanisms of STAT3 activation, we generated a series of Ba/F3 cell transfectants expressing the wild-type G-CSF-R or mutant receptors that either completely lack tyrosines or retain just one of the four cytoplasmic tyrosines of the G-CSF-R. We show that, at saturating G-CSF concentrations, STAT3 activation from the full-length G-CSF-R is efficiently mediated by the C-terminal domain in a manner independent of receptor tyrosines. In contrast, at low G-CSF concentrations, Y704 and Y744 of the G-CSF-R play a major role in STAT3 activation. Both tyrosine-dependent and -independent mechanisms of STAT3 activation are sensitive to the Jak2 inhibitor AG-490, follow similar kinetics, and lead to transactivation of a STAT3 reporter construct, indicating functional equivalence. STAT3 activation is also impaired, particularly at nonsaturating G-CSF concentrations, in bone marrow cells from mice expressing a truncated G-CSF-R (gcsfr-triangle up715). These findings suggest that G-CSF-induced STAT3 activation during basal granulopoiesis (low G-CSF) and "emergency" granulopoiesis (high G-CSF) are differentially controlled. In addition, the data establish the importance of the G-CSF-R C-terminus in STAT3 activation in primary cells, which has implications for understanding why truncated G-CSF-R derived from SCN patients are defective in maturation signaling.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fator Estimulador de Colônias de Granulócitos/fisiologia , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Tirosina/fisiologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Neutropenia/genética , Neutropenia/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/biossíntese , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Fator de Transcrição STAT3 , Transfecção , Tirosina/metabolismoRESUMO
The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF-mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF-containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF-mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF-driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Transdução de Sinais/fisiologia , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Genes myc , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/citologia , Humanos , Mutagênese Sítio-Dirigida , Neutrófilos/citologia , Receptores de Fator Estimulador de Colônias de Granulócitos/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Tirosina/químicaRESUMO
Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) involves the activation of the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway. G-CSF induces tyrosine phosphorylation of Jak1, Jak2, STAT1, and STAT3. The membrane-proximal region of G-CSF-R is sufficient for activation of Jaks. It is still unclear how STAT proteins are activated by G-CSF-R. We investigated the possible involvement of the C-terminal region of G-CSF-R in the recruitment of STAT proteins using BAF3 cell transfectants expressing wild type (WT) G-CSF-R, C-terminal deletion mutants and tyrosine-to-phenylalanine substitution mutants. Electrophoretic mobility shift assays with STAT-binding oligonucleotides (m67) showed that activation of WT G-CSF-R induces three distinct STAT complexes, namely STAT3 homodimers, STAT1-STAT3 heterodimers, and STAT1 homodimers. However, STAT1 homodimers and STAT1-STAT3 heterodimers were predominantly formed after activation of a C-terminal deletion mutant d685, which lacks all four conserved cytoplasmic tyrosine residues, located at positions 704, 729, 744, and 764. Antiphosphotyrosine immunoblots of STAT3 immunoprecipitates showed that activation of WT G-CSF-R induced phosphorylation of STAT3. In contrast, no phosphorylation of STAT3 was observed after activation of deletion mutant d685. These findings establish that the C-terminal region of G-CSF-R plays a major role in the activation of STAT3. By using tyrosine-to-phenylalanine substitution mutants of G-CSF-R, we further show that tyrosine 704, present in a YXXQ consensus sequence shown to be essential for STAT3 binding to gp130, is not exclusively involved in the activation of STAT3 by G-CSF-R.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Transativadores/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Sequência Consenso , Citoplasma , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Fosfotirosina/metabolismo , Ligação Proteica , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transdução de SinaisRESUMO
Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) occurs via multiple pathways, one of which involves activation of p21Ras and mitogen-activated protein kinase. The SH2 domain-containing proteins Shc and GRB2 have been implicated in this latter signaling route. We studied the role of these proteins in signal transduction from wild type (WT) G-CSF-R, C-terminal deletion mutants, and tyrosine-to-phenylalanine substitution mutants in transfectants of the mouse pro-B cell line, BAF3. G-CSF stimulation of BAF3 cells expressing WT G-CSF-R induced tyrosine phosphorylation of Shc. Anti-Shc antibodies co-immunoprecipitated tyrosine-phosphorylated 145-kD proteins (p145), whereas GRB2 immunoprecipitates contained phosphorylated Shc, Syp, and proteins of 145 and 90 kD (p90). Neither of these complexes were detected after activation of a C-terminal deletion mutant of G-CSF-R that lacked all four conserved cytoplasmic tyrosine residues. G-CSF induced formation of Syp/GRB2 complexes in all the tyrosine-substitution mutants, suggesting that this association did not depend on the presence of single specific tyrosine residues in G-CSF-R. In contrast, tyrosine 764 of G-CSF-R appeared to be exclusively required for tyrosine phosphorylation of Shc and its association with p145 and GRB2. In addition, tyrosine 764 also specifically mediated binding of GRB2 to p90 without the involvement of Shc. These findings indicate that tyrosine 764 of G-CSF-R has a prominent role in G-CSF signal transduction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Fator Estimulador de Colônias de Granulócitos/fisiologia , Fosfoproteínas/fisiologia , Proteínas/fisiologia , Receptores de Fator Estimulador de Colônias de Granulócitos/química , Transdução de Sinais/fisiologia , Tirosina/química , Animais , Sequência de Bases , Proteína Adaptadora GRB2 , Humanos , Substâncias Macromoleculares , Camundongos , Mitose/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Transfecção , Domínios de Homologia de srcRESUMO
Severe congenital neutropenia (Kostmann syndrome) is characterized by profound absolute neutropenia and a maturation arrest of marrow progenitor cells at the promyelocyte-myelocyte stage. Marrow cells from such patients frequently display a reduced responsiveness to granulocyte-colony-stimulating factor (G-CSF). G-CSF binds to and activates a specific receptor which transduces signals critical for the proliferation and maturation of granulocytic progenitor cells. Here we report the identification of a somatic point mutation in one allele of the G-CSF receptor gene in a patient with severe congenital neutropenia. The mutation results in a cytoplasmic truncation of the receptor. When expressed in murine myeloid cells, the mutant receptor transduced a strong growth signal but, in contrast to the wild-type G-CSF receptor, was defective in maturation induction. The mutant receptor chain may act in a dominant negative manner to block granulocytic maturation.