RESUMEN
Transfusion of donor-derived red blood cells (RBC) is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units, especially for chronically transfused patients. In vitro cultured, customizable RBC would negate these concerns and further increase precision medicine. Large-scale, cost-effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to good manufacturing practice (GMP) culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD34+ isolation, and a 3 × 107-fold increase in erythroblasts in 25 days (or from 100 million peripheral blood mononuclear cells, 2 to 4 mL packed red cells can be produced). Expanded erythroblast cultures could be differentiated to CD71dimCD235a+CD44+CD117-DRAQ5- RBC in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen-binding capacity of cultured RBC was comparable to in vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-sequencing provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1 L per reactor, allowing transition toward clinical studies and small-scale applications.
Asunto(s)
Técnicas de Cultivo Celular por Lotes , Diferenciación Celular , Eritroblastos/citología , Eritrocitos/citología , Eritropoyesis , Leucocitos Mononucleares/citología , Técnicas de Cultivo Celular por Lotes/normas , Biomarcadores , Reactores Biológicos , Diferenciación Celular/genética , Proliferación Celular , Eritroblastos/metabolismo , Eritrocitos/metabolismo , Eritropoyesis/genética , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Leucocitos Mononucleares/metabolismo , Cultivo Primario de Células , Reticulocitos/metabolismo , TranscriptomaRESUMEN
A 4-year-old boy, a ß-thalassemia (ß-thal) carrier, with an unexplained severe chronic microcytic anemia was referred to us. Sequencing of the α-globin genes revealed a Hb Charlieu [α106(G13)LeuâPro, HBA1: c.320T>C, p.Leu107Pro] mutation present on both HBA1 genes. Quantitative polymerase chain reaction (qPCR) confirmed αCharlieu mRNA in the proband and his parents, showing that the mutation does not affect mRNA stability. However, we were unable to detect the Hb Charlieu protein by capillary electrophoresis (CE), reverse phase electrophoresis, cation exchange electrophoresis or isoelectric focusing. Mass spectrometry (MS) allowed us to confirm the presence of the Hb Charlieu peptide in erythrocyte progenitors. These findings suggest that the mutation affects the stability of αCharlieu. As hemoglobin (Hb) heat stability tests showed no abnormalities in erythrocytes, we speculated that αCharlieu is already degraded during red blood cell (RBC) development. The clinical severity in the proband and the presence of new methylene blue-stained aggregates in his reticulocytes indicates that incorporation of αCharlieu destabilizes Hb. This, combined with an excess of unstable free α-globins as the result of ß-thal minor, results in severely impaired erythropoiesis and, as a consequence, severe and chronic microcytic anemia in the proband.
Asunto(s)
Homocigoto , Mutación/genética , Preescolar , Humanos , MasculinoRESUMEN
Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein α (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome.
Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Células Madre Hematopoyéticas/metabolismo , Células Mieloides/citología , Células Mieloides/metabolismo , Neutropenia/genética , Proteínas/genética , Animales , Apoptosis/genética , Enfermedades de la Médula Ósea/genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Ciclo Celular/genética , Modelos Animales de Enfermedad , Insuficiencia Pancreática Exocrina/genética , Eliminación de Gen , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Lipomatosis/genética , Ratones , Ratones Noqueados , Síndrome de Shwachman-Diamond , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Severe congenital neutropenia (CN) is a preleukemic bone marrow failure syndrome with a 20% risk of evolving into leukemia or myelodysplastic syndrome (MDS). Patterns of acquisition of leukemia-associated mutations were investigated using next-generation deep-sequencing in 31 CN patients who developed leukemia or MDS. Twenty (64.5%) of the 31 patients had mutations in RUNX1. A majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R mutations. In contrast to their high frequency in CN patients who developed leukemia or MDS, RUNX1 mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric acute myeloid leukemia. A sequential analysis at stages prior to overt leukemia revealed RUNX1 mutations to be late events in leukemic transformation. Single-cell analyses in 2 patients showed that RUNX1 and CSF3R mutations were present in the same malignant clone. Functional studies demonstrated elevated granulocyte colony-stimulating factor (G-CSF)-induced proliferation with diminished myeloid differentiation of hematopoietic CD34(+) cells coexpressing mutated forms of RUNX1 and CSF3R. The high frequency of cooperating RUNX1 and CSF3R mutations in CN patients suggests a novel molecular pathway of leukemogenesis: mutations in the hematopoietic cytokine receptor (G-CSFR) in combination with the second mutations in the downstream hematopoietic transcription fator (RUNX1). The detection of both RUNX1 and CSF3R mutations could be used as a marker for identifying CN patients with a high risk of progressing to leukemia or MDS.
Asunto(s)
Transformación Celular Neoplásica/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Leucemia Mieloide/genética , Mutación , Neutropenia/congénito , Receptores del Factor Estimulante de Colonias/genética , Adolescente , Adulto , Niño , Preescolar , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Análisis Citogenético , Femenino , Humanos , Masculino , Neutropenia/genética , Neutropenia/patología , Transducción de Señal/genética , Adulto JovenRESUMEN
The endonuclease complex Ercc1/Xpf is involved in interstrand crosslink repair and functions downstream of the Fanconi pathway. Loss of Ercc1 causes hematopoietic defects similar to those seen in Fanconi Anemia. Ercc1(-/-) mice die 3-4 weeks after birth, which prevents long-term follow up of the hematopoietic compartment. We used alternative Ercc1 mouse models to examine the effect of low or absent Ercc1 activity on hematopoiesis. Tie2-Cre-driven deletion of a floxed Ercc1 allele was efficient (>80%) in fetal liver hematopoietic cells. Hematopoietic stem and progenitor cells (HSPCs) with a deleted allele were maintained in mice up to 1 year of age when harboring a wt allele, but were progressively outcompeted when the deleted allele was combined with a knockout allele. Mice with a minimal Ercc1 activity expressed by 1 or 2 hypomorphic Ercc1 alleles have an extended life expectancy, which allows analysis of HSPCs at 10 and 20 weeks of age. The HSPC compartment was affected in all Ercc1-deficient models. Actively proliferating multipotent progenitors were most affected as were myeloid and erythroid clonogenic progenitors. In conclusion, lack of Ercc1 results in a severe competitive disadvantage of HSPCs and is most deleterious in proliferating progenitor cells.
RESUMEN
Severe congenital neutropenia (SCN) is a BM failure syndrome with a high risk of progression to acute myeloid leukemia (AML). The underlying genetic changes involved in SCN evolution to AML are largely unknown. We obtained serial hematopoietic samples from an SCN patient who developed AML 17 years after the initiation of G-CSF treatment. Next- generation sequencing was performed to identify mutations during disease progression. In the AML phase, we found 12 acquired nonsynonymous mutations. Three of these, in CSF3R, LLGL2, and ZC3H18, co-occurred in a subpopulation of progenitor cells already in the early SCN phase. This population expanded over time, whereas clones harboring only CSF3R mutations disappeared from the BM. The other 9 mutations were only apparent in the AML cells and affected known AML-associated genes (RUNX1 and ASXL1) and chromatin remodelers (SUZ12 and EP300). In addition, a novel CSF3R mutation that conferred autonomous proliferation to myeloid progenitors was found. We conclude that progression from SCN to AML is a multistep process, with distinct mutations arising early during the SCN phase and others later in AML development. The sequential gain of 2 CSF3R mutations implicates abnormal G-CSF signaling as a driver of leukemic transformation in this case of SCN.
Asunto(s)
Transformación Celular Neoplásica/genética , Leucemia Mieloide Aguda/genética , Mutación , Proteínas de Neoplasias/genética , Neutropenia/genética , Adulto , Médula Ósea/metabolismo , Transformación Celular Neoplásica/metabolismo , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Humanos , Leucemia Mieloide Aguda/complicaciones , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Proteínas de Neoplasias/metabolismo , Neutropenia/complicaciones , Neutropenia/congénito , Neutropenia/tratamiento farmacológico , Neutropenia/metabolismo , Células Madre/metabolismoRESUMEN
Steady-state hematopoiesis is altered on infection, but the cellular and molecular mechanisms driving these changes are largely unknown. Modulation of hematopoiesis is essential to increase the output of the appropriate type of effector cell required to combat the invading pathogen. In the present study, we demonstrate that the pro-inflammatory cytokine IFNγ is involved in orchestrating inflammation-induced myelopoiesis. Using both mouse models and in vitro assays, we show that IFNγ induces the differentiation of monocytes over neutrophils at the level of myeloid progenitors. Infection with lymphocytic choriomeningitis virus induces monopoiesis in wild-type mice, but causes increased neutrophil production in IFNγ(-/-) mice. We demonstrate that IFNγ enhances the expression of the monopoiesis-inducing transcription factors IRF8 and PU.1 in myeloid progenitor cells, whereas it reduces G-CSF-driven neutrophil differentiation via a SOCS3-dependent inhibition of STAT3 phosphorylation. These results establish a critical role for IFNγ in directing monocyte versus neutrophil development during immune activation.
Asunto(s)
Mediadores de Inflamación/fisiología , Inflamación/metabolismo , Interferón gamma/fisiología , Neutrófilos/metabolismo , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Ligando CD27/genética , Ligando CD27/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos/farmacología , Inflamación/genética , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/farmacología , Factores Reguladores del Interferón/metabolismo , Interferón gamma/genética , Interferón gamma/farmacología , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Monocitos/citología , Monocitos/metabolismo , Células Progenitoras Mieloides/metabolismo , Mielopoyesis/efectos de los fármacos , Neutrófilos/citología , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transactivadores/metabolismoRESUMEN
Acute myeloid leukemia (AML) results from multiple genetic and epigenetic aberrations, many of which remain unidentified. Frequent loss of large chromosomal regions marks haplo-insufficiency as one of the major mechanisms contributing to leukemogenesis. However, which haplo-insufficient genes (HIGs) are involved in leukemogenesis is largely unknown and powerful experimental strategies aimed at their identification are currently lacking. Here, we present a new approach to discover HIGs, using retroviral integration mutagenesis in mice in which methylated viral integration sites and neighbouring genes were identified. In total we mapped 6 genes which are flanked by methylated viral integration sites (mVIS). Three of these, i.e., Lrmp, Hcls1 and Prkrir, were up regulated and one, i.e., Ptp4a3, was down regulated in the affected tumor. Next, we investigated the role of PTP4A3 in human AML and we show that PTP4A3 expression is a negative prognostic indicator, independent of other prognostic parameters. In conclusion, our novel strategy has identified PTP4A3 to potentially have a role in AML, on one hand as a candidate HIG contributing to leukemogenesis in mice and on the other hand as a prognostic indicator in human AML.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Virus de la Leucemia Murina/fisiología , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Mutagénesis , Proteínas de Neoplasias/genética , Proteínas Tirosina Fosfatasas/genética , Integración Viral/genética , Animales , Metilación de ADN/genética , Regulación hacia Abajo/genética , Haploinsuficiencia/genética , Humanos , Virus de la Leucemia Murina/genética , Leucemia Mieloide Aguda/virología , Ratones , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pronóstico , Transcripción Genética/genéticaRESUMEN
Hematopoiesis is tightly controlled by transcription regulatory networks, but how and when specific transcription factors control lineage commitment are still largely unknown. Within the hematopoietic stem cell (Lin(-)Sca-1(+)c-Kit(+)) compartment these lineage-specific transcription factors are expressed at low levels but are up-regulated with the process of lineage specification. CCAAT/enhancer binding protein α (C/EBPα) represents one of these factors and is involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor cells, which express C/EBPα, we developed a mouse model expressing Cre recombinase from the Cebpa promoter and a conditional EYFP allele. We show that Cebpa/EYFP(+) cells represent a significant subset of multipotent hematopoietic progenitors, which predominantly give rise to myeloid cells in steady-state hematopoiesis. C/EBPα induced a strong myeloid gene expression signature and down-regulated E2A-induced regulators of early lymphoid development. In addition, Cebpa/EYFP(+) cells compose a fraction of early thymic progenitors with robust myeloid potential. However, Cebpa/EYFP(+) multipotent hematopoietic progenitors and early thymic progenitors retained the ability to develop into erythroid and T-lymphoid lineages, respectively. These findings support an instructive but argue against a lineage-restrictive role of C/EBPα in multipotent hematopoietic and thymic progenitors.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Linaje de la Célula , Células Madre Hematopoyéticas/citología , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Timo/citología , Timo/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Compartimento Celular , Proliferación Celular , Ensayo de Unidades Formadoras de Colonias , Células Dendríticas/citología , Células Dendríticas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Integrasas/metabolismo , Proteínas Luminiscentes/metabolismo , Ratones , Células Mieloides/citología , Células Mieloides/metabolismo , Técnicas de Cultivo de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo/embriologíaRESUMEN
Thioredoxin-interacting protein (TXNIP) is involved in reactive oxygen species-induced stress responses. In a screen for novel disease genes in murine leukemia virus (MLV)-induced mouse leukemias, we identified Txnip as a frequent target for proviral integration. Ectopic TXNIP expression inhibited the proliferation of myeloid progenitor cells. TXNIP transcript and protein levels were significantly elevated in human AML blasts of certain patients, particularly those harboring translocation t(8;21). Nucleotide sequencing revealed no abnormalities in the TXNIP coding region in AML. These findings suggest that deregulated TXNIP expression contributes to MLV-induced murine leukemia as well as human AML.
Asunto(s)
Proteínas Portadoras/genética , Regulación de la Expresión Génica , Leucemia Experimental/genética , Leucemia Mieloide Aguda/genética , Leucemia/genética , Tiorredoxinas/genética , Integración Viral/genética , Animales , Crisis Blástica/genética , Crisis Blástica/patología , Línea Celular , Cartilla de ADN , Genes Reporteros , Humanos , Virus de la Leucemia Murina/genética , Leucemia Mieloide Aguda/patología , Ratones , Reacción en Cadena de la Polimerasa , Regiones Promotoras GenéticasRESUMEN
CSF3R [G-CSF (granulocyte colony-stimulating factor) receptor] controls survival, proliferation and differentiation of myeloid progenitor cells via activation of multiple JAKs (Janus kinases). In addition to their role in phosphorylation of receptor tyrosine residues and downstream signalling substrates, JAKs have recently been implicated in controlling expression of cytokine receptors, predominantly by masking critical motifs involved in endocytosis and lysosomal targeting. In the present study, we show that increasing the levels of JAK1, JAK2 and TYK2 (tyrosine kinase 2) elevated steady-state CSF3R cell-surface expression and enhanced CSF3R protein stability in haematopoietic cells. This effect was not due to inhibition of endocytotic routing, since JAKs did not functionally interfere with the dileucine-based internalization motif or lysine-mediated lysosomal degradation of CSF3R. Rather, JAKs appeared to act on CSF3R in the biosynthetic pathway at the level of the ER (endoplasmic reticulum). Strikingly, increased JAK levels synergized with internalization- or lysosomal-routing-defective CSF3R mutants to confer growth-factor independent STAT3 (signal transducer and activator of transcription 3) activation and cell survival, providing a model for how increased JAK expression and disturbed intracellular routing of CSF3R synergize in the transformation of haematopoietic cells.
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Membrana Celular/metabolismo , Quinasas Janus/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Endocitosis , Células HeLa , Humanos , Ligandos , Lisosomas/metabolismo , Ratones , Receptores del Factor Estimulante de Colonias/genética , Receptores del Factor Estimulante de Colonias/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Transfección , UbiquitinaciónRESUMEN
The granulocyte colony-stimulating factor receptor (G-CSF-R) transmits signals for proliferation and differentiation of myeloid progenitor cells. Here we report on the identification of a rare single nucleotide polymorphism within its intracellular domain (G-CSF-R_Glu785Lys). Screening a cohort of 116 patients with primary myelodysplastic syndromes (MDS), de novo acute myeloid leukemia (AML) (84 patients), as well as 232 age- and sex-matched controls revealed a highly significant association of the G-CSF-R_785Lys allele with the development of high-risk MDS as defined by more than 5% bone marrow blasts (9.7% versus 0.9% in controls; P = .001; odds ratio [OR], 12.5; 95% confidence interval [CI], 2.4-58.9) or an International Prognostic Scoring System score of intermediate-2 or high (13.0% versus 0.9%; P < .001; OR, 14.0; 95% CI, 3.4-85.0). Functional analysis by retroviral transfer of G-CSF-R_785Lys into myeloid progenitor cells of G-CSF-R-deficient mice showed a significantly diminished colony-formation capacity after G-CSF stimulation as compared with cells transduced with the wild-type receptor. These results suggest that lifelong altered G-CSF response by the G-CSF-R_785Lys may render individuals susceptible to development of high-risk MDS.
Asunto(s)
Síndromes Mielodisplásicos/genética , Polimorfismo de Nucleótido Simple , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Animales , Estudios de Casos y Controles , Células Cultivadas , Femenino , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Células Madre Hematopoyéticas , Humanos , Leucemia Mieloide/genética , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Síndromes Mielodisplásicos/etiología , Síndromes Mielodisplásicos/patología , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/trasplante , Riesgo , Transducción Genética , TransfecciónRESUMEN
Acute myeloid leukemia (AML) is a heterogeneous group of diseases in which chromosomal aberrations, small insertions or deletions, or point mutations in certain genes have profound consequences for prognosis. However, the majority of AML patients present without currently known genetic defects. Retroviral insertion mutagenesis in mice has become a powerful tool for identifying new disease genes involved in the pathogenesis of leukemia and lymphoma. Here we have used the Graffi-1.4 strain of murine leukemia virus, which causes predominantly AML, in a screen to identify novel genes involved in the pathogenesis of this disease. We report 79 candidate disease genes in common integration sites (CISs) and 15 genes whose family members previously were found to be affected in other studies. The majority of the identified sequences (60%) were not found in lymphomas and monocytic leukemias in previous screens, suggesting a specific involvement in AML. Although most of the virus integrations occurred in or near the 5' or 3' ends of the genes, suggesting deregulation of gene expression as a consequence of virus integration, 18 CISs were located exclusively within the genes, conceivably causing gene disruption.
Asunto(s)
Virus de la Leucemia Murina/patogenicidad , Leucemia Mieloide/genética , Proteínas/genética , Enfermedad Aguda , Animales , Animales Recién Nacidos , Leucemia Experimental/genética , Leucemia Experimental/fisiopatología , Leucemia Experimental/virología , Leucemia Mieloide/fisiopatología , Ratones , Mutagénesis Insercional , Provirus/genética , Infecciones por Retroviridae/virología , Análisis de Secuencia de ADN , Infecciones Tumorales por Virus/virología , Integración ViralRESUMEN
The genetic defects underlying the pathogenesis of acute myeloid leukemia (AML) are still largely unknown. Retroviral insertion mutagenesis in mice has become a powerful tool to identify candidate genes involved in the development of leukemia and lymphoma. We have used this strategy with the 1.4 strain of Graffi murine leukemia virus (MuLV), which predominantly causes myeloid leukemias. Here, we report that Graffi-1.4-induced AML frequently harbors virus integrations in the gene encoding the transcription factor Yin Yang 1 (YY1). These integrations occurred in both orientations, and all were located in the 5' promoter region of the gene, 0.5 to 1.5 kb upstream of the major transcriptional start site. Luciferase reporter assays showed that virus integration in this region increases promoter activity and renders it independent of a functional binding site for Sp1, a major transcriptional regulator of YY1. We used the murine 32D model to study the consequence of perturbed YY1 expression for myelopoiesis. YY1 protein levels were high in 32D parental cells maintained in interleukin-3-containing medium, but they dropped when the cells were induced to differentiate by granulocyte-colony-stimulating factor (G-CSF). Strikingly, G-CSF-induced neutrophilic differentiation was reduced in 32D cell transfectants ectopically expressing YY1. In similar experiments on primary bone marrow cells, enforced YY1 expression blocked the outgrowth of CFU-GM colonies. Increased YY1 expression was seen in some cases of human AML. Collectively, these data imply a possible role of perturbed expression of YY1 in the development of AML through interference with the myeloid differentiation program in the leukemic progenitor cells.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/genética , Leucemia Mieloide/genética , Neutrófilos/patología , Factores de Transcripción/genética , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Transformación Celular Viral/genética , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Regulación Leucémica de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos/farmacología , Humanos , Virus de la Leucemia Murina , Leucemia Mieloide/etiología , Leucemia Mieloide/virología , Ratones , Mielopoyesis/genética , Oncogenes/genética , Oncogenes/fisiología , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Factores de Transcripción/metabolismo , Integración Viral/genética , Integración Viral/fisiología , Factor de Transcripción YY1RESUMEN
Mycophenolic acid (MPA) and methotrexate (MTX) are immunosuppressive drugs used for the treatment of various immunological disorders. MPA is an inhibitor of inosine monophosphate dehydrogenase and MTX is a folate antagonist that inhibits tetrahydrofolate reductase. Production of T cell cytokines in whole blood cultures, as well as in PBMC cultures, is inhibited by a low concentration of both drugs. Inhibition of cytokine production after monocyte stimulation was less evident. The mechanism by which inhibition is achieved is different for both drugs. Inhibition of T cell cytokine production by MPA was more profound and started earlier compared to the inhibition by MTX. MTX induced apoptosis in T cells that became activated, whereas MPA prevented activation of T cells by arresting the cell cycle in the G0/G1 phase. Addition of guanosine and adenosine can overcome this cell cycle arrest, even after several days. Furthermore MPA inhibited the expression of activation markers HLA-DR and CD71 on T cells. The observation that MTX cannot prevent T cell activation but induces apoptosis in activated T cells, and that MPA reversibly prevents activation of T cells could explain the immunosuppressive effects of both these drugs.