RESUMEN
Human red cell differentiation requires the action of erythropoietin on committed progenitor cells. In iron deficiency, committed erythroid progenitors lose responsiveness to erythropoietin, resulting in hypoplastic anemia. To address the basis for iron regulation of erythropoiesis, we established primary hematopoietic cultures with transferrin saturation levels that restricted erythropoiesis but permitted granulopoiesis and megakaryopoiesis. Experiments in this system identified as a critical regulatory element the aconitases, multifunctional iron-sulfur cluster proteins that metabolize citrate to isocitrate. Iron restriction suppressed mitochondrial and cytosolic aconitase activity in erythroid but not granulocytic or megakaryocytic progenitors. An active site aconitase inhibitor, fluorocitrate, blocked erythroid differentiation in a manner similar to iron deprivation. Exogenous isocitrate abrogated the erythroid iron restriction response in vitro and reversed anemia progression in iron-deprived mice. The mechanism for aconitase regulation of erythropoiesis most probably involves both production of metabolic intermediates and modulation of erythropoietin signaling. One relevant signaling pathway appeared to involve protein kinase Calpha/beta, or possibly protein kinase Cdelta, whose activities were regulated by iron, isocitrate, and erythropoietin.
Asunto(s)
Células Precursoras Eritroides/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Proteína 1 Reguladora de Hierro/metabolismo , Hierro/farmacología , Anemia Ferropénica/sangre , Anemia Ferropénica/etiología , Anemia Ferropénica/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Femenino , Citometría de Flujo , Humanos , Immunoblotting , Deficiencias de Hierro , Proteína 1 Reguladora de Hierro/genética , Isocitratos/administración & dosificación , Células K562 , Masculino , Ratones , Ratones Endogámicos C57BL , Interferencia de ARN , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: To evaluate the safety and efficacy of high-dose [(131)I]metaiodobenzylguanidine ([(131)I]MIBG) in the treatment of malignant pheochromocytoma (PHEO) and paraganglioma (PGL). METHODS: Fifty patients with metastatic PHEO or PGL, age 10 to 64 years, were treated with [(131)I]MIBG doses ranging from 492 to 1,160 mCi (median, 12 mCi/kg). Cumulative [(131)I]MIBG administered ranged from 492 to 3,191 mCi. Autologous hematopoietic stem cells were collected and cryopreserved before treatment with [(131)I]MIBG greater than 12 mCi/kg or with a total dose greater than 500 mCi. Sixty-nine [(131)I]MIBG infusions were given, which included infusions to 35 patients treated once and infusions to 15 patients who received two or three treatments. Response was evaluated by [(123)I]MIBG scans, computed tomography/magnetic resonance imaging, urinary catecholamines/metanephrines, and chromogranin A. RESULTS: The overall complete response (CR) plus partial response (PR) rate in 49 evaluable patients was 22%. Additionally, 35% of patients achieved a CR or PR in at least one measure of response without progressive disease, and 8% of patients maintained stable disease for greater than 12 months. Thirty-five percent of patients experienced progressive disease within 1 year after therapy. The estimated 5-year overall survival rate was 64%. Toxicities included grades 3 to 4 neutropenia (87%) and thrombocytopenia (83%). Grades 3 to 4 nonhematologic toxicity included acute respiratory distress syndrome (n = 2), bronchiolitis obliterans organizing pneumonia (n = 2), pulmonary embolism (n = 1), fever with neutropenia (n = 7), acute hypertension (n = 10), infection (n = 2), myelodysplastic syndrome (n = 2), and hypogonadism (n = 4). CONCLUSION: Although serious toxicity may occur, the survival and response rates achieved with high-dose [(131)I]MIBG suggest its utility in the management of selected patients with metastatic PHEO and PGL.
Asunto(s)
3-Yodobencilguanidina/administración & dosificación , Neoplasias de las Glándulas Suprarrenales/radioterapia , Radioisótopos de Yodo/administración & dosificación , Paraganglioma/radioterapia , Feocromocitoma/radioterapia , Radiofármacos/administración & dosificación , 3-Yodobencilguanidina/efectos adversos , Adolescente , Neoplasias de las Glándulas Suprarrenales/diagnóstico , Neoplasias de las Glándulas Suprarrenales/mortalidad , Neoplasias de las Glándulas Suprarrenales/secundario , Adulto , Biomarcadores de Tumor/orina , Catecolaminas/orina , Niño , Cromogranina A/orina , Supervivencia sin Enfermedad , Relación Dosis-Respuesta en la Radiación , Femenino , Humanos , Infusiones Intravenosas , Radioisótopos de Yodo/efectos adversos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Paraganglioma/diagnóstico , Paraganglioma/secundario , Feocromocitoma/diagnóstico , Feocromocitoma/mortalidad , Feocromocitoma/secundario , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Radiofármacos/efectos adversos , Medición de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
The transcription factor GATA-1 participates in programming the differentiation of multiple hematopoietic lineages. In megakaryopoiesis, loss of GATA-1 function produces complex developmental abnormalities and underlies the pathogenesis of megakaryocytic leukemia in Down syndrome. Its distinct functions in megakaryocyte and erythroid maturation remain incompletely understood. In this study, we identified functional and physical interaction of GATA-1 with components of the positive transcriptional elongation factor P-TEFb, a complex containing cyclin T1 and the cyclin-dependent kinase 9 (Cdk9). Megakaryocytic induction was associated with dynamic changes in endogenous P-TEFb composition, including recruitment of GATA-1 and dissociation of HEXIM1, a Cdk9 inhibitor. shRNA knockdowns and pharmacologic inhibition both confirmed contribution of Cdk9 activity to megakaryocytic differentiation. In mice with megakaryocytic GATA-1 deficiency, Cdk9 inhibition produced a fulminant but reversible megakaryoblastic disorder reminiscent of the transient myeloproliferative disorder of Down syndrome. P-TEFb has previously been implicated in promoting elongation of paused RNA polymerase II and in programming hypertrophic differentiation of cardiomyocytes. Our results offer evidence for P-TEFb cross-talk with GATA-1 in megakaryocytic differentiation, a program with parallels to cardiomyocyte hypertrophy.
Asunto(s)
Diferenciación Celular , Quinasa 9 Dependiente de la Ciclina/fisiología , Factor de Transcripción GATA1/metabolismo , Megacariocitos/citología , Factor B de Elongación Transcripcional Positiva/metabolismo , Receptor Cross-Talk , Animales , Células Cultivadas , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Síndrome de Down , Factor de Transcripción GATA1/genética , Humanos , Ratones , Ratones Noqueados , Trastornos MieloproliferativosRESUMEN
Coculture with stromal cells tends to maintain normal hematopoietic progenitors and their leukemic counterparts in an undifferentiated, proliferative state. An example of this effect is seen with megakaryocytic differentiation, wherein stromal contact renders many cell types refractory to potent induction stimuli. This inhibitory effect of stroma on megakaryocytic differentiation correlates with a blockade within hematopoietic cells of protein kinase C-epsilon (PKC-epsilon) up-regulation and of extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase activation, both of which have been implicated in promoting megakaryocytic differentiation. In this study K562DeltaRafER.5 cells, expressing an estradiol-responsive mutant of the protein kinase Raf-1, were used to determine the relevance and stage of ERK/MAPK pathway blockade by stromal contact. Activation of DeltaRafER by estradiol overrode stromal blockade of megakaryocytic differentiation, implicating the proximal stage of the ERK/MAPK pathway as a relevant control point. Because stromal contact blocked delayed but not early ERK activation, the small guanosine triphosphatase (GTPase) Rap1 was considered as a candidate inhibitory target. Activation assays confirmed that Rap1 underwent sustained activation as a result of megakaryocytic induction, as previously described. As with ERK activation, stromal contact selectively blocked delayed but not early Rap1 activation, having no effect on Ras activation. Enforced expression of either wild-type Rap1 or the GTPase (GAP) resistant mutant Rap1 V12 failed to override stromal inhibition, suggesting that the inhibitory mechanism does not involve GAP up-regulation but rather may target upstream guanine nucleotide exchange factor (GEF) complexes. Accordingly, coimmunoprecipitation demonstrated stromally induced alterations in a protein complex associated with c-Cbl, a scaffolding factor for Rap1-GEF complexes.