RESUMEN
Benzene, a widely used industrial chemical, has been clarified to cause hematotoxicity. Our previous study suggested that miR-451a may play a role in benzene-induced impairment of erythroid differentiation. However, the mechanism underlying remains unclear. In this study, we explored the role of miR-451a and its underlying mechanisms in hydroquinone (HQ)-induced suppression of erythroid differentiation in K562 cells. 0, 1.0, 2.5, 5.0, 10.0, and 50⯵M HQ treatment of K562 cells resulted in a dose-dependent inhibition of erythroid differentiation, as well as the expression of miR-451a. Bioinformatics analysis was conducted to predict potential target genes of miR-451a and dual-luciferase reporter assays confirmed that miR-451a can directly bind to the 3'-UTR regions of BATF, SETD5, and ARHGEF3 mRNAs. We further demonstrated that over-expression or down-regulation of miR-451a altered the expression of BATF, SETD5, and ARHGEF3, and also modified erythroid differentiation. In addition, BATF, SETD5, and ARHGEF3 were verified to play a role in HQ-induced inhibition of erythroid differentiation in this study. Knockdown of SETD5 and ARHGEF3 reversed HQ-induced suppression of erythroid differentiation while knockdown of BATF had the opposite effect. On the other hand, we also identified c-Jun as a potential transcriptional regulator of miR-451a. Forced expression of c-Jun increased miR-451a expression and reversed the inhibition of erythroid differentiation induced by HQ, whereas knockdown of c-Jun had the opposite effect. And the binding site of c-Jun and miR-451a was verified by dual-luciferase reporter assay. Collectively, our findings indicate that miR-451a and its downstream targets BATF, SETD5, and ARHGEF3 are involved in HQ-induced erythroid differentiation disorder, and c-Jun regulates miR-451a as a transcriptional regulator in this process.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Diferenciación Celular , MicroARNs , Factores de Intercambio de Guanina Nucleótido Rho , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Diferenciación Celular/efectos de los fármacos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Células K562 , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Metiltransferasas/genética , Metiltransferasas/metabolismoRESUMEN
Destruction of erythropoiesis process leads to various diseases, including thrombocytopenia, anaemia, and leukaemia. miR-429-CT10 regulation of kinase-like (CRKL) axis involved in development, progression and metastasis of cancers. However, the exact role of miR-429-CRKL axis in leukaemic cell differentiation are still unknown. The current work aimed to uncover the effect of miR-429-CRKL axis on erythropoiesis. In the present study, CRKL upregulation was negatively correlated with miR-429 downregulation in both chronic myeloid leukaemia (CML) patient and CR patient samples. Moreover, CRKL expression level was significantly decreased while miR-429 expression level was increased during the erythroid differentiation of K562 cells following hemin treatment. Functional investigations revealed that overexpression and knockdown of CRKL was remarkably effective in suppressing and promoting hemin-induced erythroid differentiation of K562 cells, whereas, miR-429 exhibited opposite effects to CRKL. Mechanistically, miR-429 regulates erythroid differentiation of K562 cells by downregulating CRKL via selectively targeting CRKL-3'-untranslated region (UTR) through Raf/MEK/ERK pathway. Conversely, CRKII had no effect on erythroid differentiation of K562 cells. Taken together, our data demonstrated that CRKL (but not CRKII) and miR-429 contribute to development, progression and erythropoiesis of CML, miR-429-CRKL axis regulates erythropoiesis of K562 cells via Raf/MEK/ERK pathway, providing novel insights into effective diagnosis and therapy for CML patients.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Diferenciación Celular , Células Eritroides , Hemina , Leucemia Mielógena Crónica BCR-ABL Positiva , MicroARNs , Proteínas Proto-Oncogénicas c-crk , Humanos , Regiones no Traducidas 3' , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Diferenciación Celular/efectos de los fármacos , Células Eritroides/metabolismo , Células Eritroides/efectos de los fármacos , Células Eritroides/patología , Células Eritroides/citología , Eritropoyesis/genética , Eritropoyesis/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Hemina/farmacología , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-crk/metabolismo , Proteínas Proto-Oncogénicas c-crk/genéticaRESUMEN
Sickle cell disease (SCD) is the most common ß-hemoglobinopathy caused by various mutations in the adult ß-globin gene resulting in sickle hemoglobin production, chronic hemolytic anemia, pain, and progressive organ damage. The best therapeutic strategies to manage the clinical symptoms of SCD is the induction of fetal hemoglobin (HbF) using chemical agents. At present, among the Food and Drug Administration-approved drugs to treat SCD, hydroxyurea is the only one proven to induce HbF protein synthesis, however, it is not effective in all people. Therefore, we evaluated the ability of the novel Bach1 inhibitor, HPP-D to induce HbF in KU812 cells and primary sickle erythroid progenitors. HPP-D increased HbF and decreased Bach1 protein levels in both cell types. Furthermore, chromatin immunoprecipitation assay showed reduced Bach1 and increased NRF2 binding to the γ-globin promoter antioxidant response elements. We also observed increased levels of the active histone marks H3K4Me1 and H3K4Me3 supporting an open chromatin configuration. In primary sickle erythroid progenitors, HPP-D increased γ-globin transcription and HbF positive cells and reduced sickled erythroid progenitors under hypoxia conditions. Collectively, our data demonstrate that HPP-D induces γ-globin gene transcription through Bach1 inhibition and enhanced NRF2 binding in the γ-globin promoter antioxidant response elements.
Asunto(s)
Anemia de Células Falciformes , gamma-Globinas , Humanos , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , gamma-Globinas/genética , Hemoglobina Falciforme/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/uso terapéutico , Activación Transcripcional/efectos de los fármacos , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismoRESUMEN
Reversal of fetal hemoglobin (HbF) silencing is an attractive therapeutic intervention for ß-thalassemia and sickle cell anemia. The current study proposes the therapeutic of repurposing of cilostazol, an FDA-approved antithrombotic agent, as a promising HbF inducer. Preliminary, we report that cilostazol induced erythroid differentiation and hemoglobinization of human erythroleukemia K562 cells. The erythroid differentiation was accompanied by increased expression of γ-globin mRNA transcripts and HbF production. Cilostazol induced erythroid differentiation and HbF production, without significantly affecting proliferation and viability of hemoglobin producing cells at maximum erythroid inducing concentration. Moreover, we investigated the effect of cilostazol on human ß- and γ-globin transgenes in in vivo ß-YAC transgenic mice, harboring human ß-locus along with ß-LCR. A good in vitro correlation was found with substantial up-regulation in fetal globin mRNA; whereas, the ß-globin gene expression was not significantly changed. F-cells, analysis in the peripheral blood of cilostazol-treated mice, revealed a significant increase in the F-cells population as compared with sham control groups. Together, these findings support the potential of cilostazol as an HbF inducer, which can be evaluated further to develop a new HbF inducer.
Asunto(s)
Cilostazol/farmacología , Hemoglobina Fetal/biosíntesis , Hemoglobinopatías/tratamiento farmacológico , Globinas beta/metabolismo , gamma-Globinas/metabolismo , Anemia de Células Falciformes/tratamiento farmacológico , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cilostazol/uso terapéutico , Reposicionamiento de Medicamentos , Células Eritroides/efectos de los fármacos , Hemoglobina Fetal/efectos de los fármacos , Hemoglobina Fetal/genética , Hemoglobinas/efectos de los fármacos , Hemoglobinas/metabolismo , Humanos , Células K562 , Ratones Transgénicos , Globinas beta/genética , Talasemia beta/tratamiento farmacológico , gamma-Globinas/genéticaRESUMEN
GATA binding protein 1 (GATA1) is one of the most important hematopoietic transcription factors in the production of blood cells, such as platelets, eosinophils, mast cells and erythrocytes. GATA1 regulates the participation of microRNA (miRNAs/miRs) in erythroid differentiation under normoxia. However, GATA1 expression and the regulation of miR2103p in the context of erythroid differentiation under hypoxia remain unknown. The present study examined the expression levels of GATA1 and miR2103p in the model of erythroid differentiation in K562 cells under hypoxia, and determined the effects of GATA1, miR2103p and SMAD2 on erythroid differentiation through lentivirus transfection experiments. The present study detected increased GATA1 expression under hypoxia. Moreover, miR2103p was identified as a positive regulator of erythroid differentiation, which was upregulated both during erythroid differentiation and in GATA1 overexpression experiments under hypoxia. Importantly, in the K562 cell model of erythroid differentiation under hypoxia, miR2103p was upregulated in a GATA1dependent manner. Using a double luciferase reporter assay, miR2103p was identified as a downstream target of GATA1mediated regulation of erythropoiesis. Gain or lossoffunction analysis of miR2103p identified its importance in erythroid differentiation. Furthermore, it was found that SMAD2 may be a downstream target gene for miR2103p. Bioinformatics predictions suggested that SMAD2 mediated miR2103pinduced regulation of erythroid differentiation. Collectively, the present study provides novel insights into the miRNA regulation of erythroid differentiation.
Asunto(s)
Hipoxia de la Célula/genética , Células Eritroides/metabolismo , MicroARNs/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Eritroides/citología , Células Eritroides/efectos de los fármacos , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Técnicas de Silenciamiento del Gen , Hemina/farmacología , Humanos , Células K562 , MicroARNs/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Immortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Eritrocitos/metabolismo , Células Eritroides/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Inhibidores de Histona Desacetilasas/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Eritrocitos/citología , Células Eritroides/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Histona Desacetilasas/aislamiento & purificación , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by clonal expansion of abnormal hematopoietic stem cells leading to hyperproliferation of one or more myeloid lineages. The main complications in MPNs are high risk of thrombosis and progression to myelofibrosis and leukemia. MPN patients with high risk scores are treated by hydroxyurea (HU), interferon-α, or ruxolitinib, a tyrosine kinase inhibitor. Polycythemia vera (PV) is an MPN characterized by overproduction of red blood cells (RBCs). ABCG2 is a member of the ATP-binding cassette superfamily transporters known to play a crucial role in multidrug resistance development. Proteome analysis showed higher ABCG2 levels in PV RBCs compared to RBCs from healthy controls and an additional increase of these levels in PV patients treated with HU, suggesting that ABCG2 might play a role in multidrug resistance in MPNs. In this work, we explored the role of ABCG2 in the transport of ruxolitinib and HU using human cell lines, RBCs, and in vitro differentiated erythroid progenitors. Using stopped-flow analysis, we showed that HU is not a substrate for ABCG2. Using transfected K562 cells expressing three different levels of recombinant ABCG2, MPN RBCs, and cultured erythroblasts, we showed that ABCG2 potentiates ruxolitinib-induced cytotoxicity that was blocked by the ABCG2-specific inhibitor KO143 suggesting ruxolitinib intracellular import by ABCG2. In silico modeling analysis identified possible ruxolitinib-binding site locations within the cavities of ABCG2. Our study opens new perspectives in ruxolitinib efficacy research targeting cell types depending on ABCG2 expression and polymorphisms among patients.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Eritrocitos/metabolismo , Proteínas de Neoplasias/metabolismo , Policitemia Vera/tratamiento farmacológico , Pirazoles/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/química , Apoptosis/efectos de los fármacos , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Línea Celular , Simulación por Computador , Dicetopiperazinas/farmacología , Eritrocitos/efectos de los fármacos , Células Eritroides/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Hidroxiurea/metabolismo , Hidroxiurea/farmacología , Interferón-alfa/farmacología , Células K562 , Trastornos Mieloproliferativos/sangre , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Nitrilos , Fosfatidilserinas/metabolismo , Policitemia Vera/sangre , Policitemia Vera/patología , Pirazoles/química , Pirazoles/metabolismo , Pirazoles/farmacocinética , PirimidinasRESUMEN
Diamond Blackfan Anemia (DBA) is a congenital macrocytic anemia associated with ribosomal protein haploinsufficiency. Ribosomal dysfunction delays globin synthesis, resulting in excess toxic free heme in erythroid progenitors, early differentiation arrest, and pure red cell aplasia. In this study, DBA induced pluripotent stem cell (iPSC) lines were generated from blood mononuclear cells of DBA patients with inactivating mutations in RPS19 and subjected to hematopoietic differentiation to model disease phenotypes. In vitro differentiated hematopoietic cells were used to investigate whether eltrombopag, an FDA-approved mimetic of thrombopoietin with robust intracellular iron chelating properties, could rescue erythropoiesis in DBA by restricting the labile iron pool (LIP) derived from excessive free heme. DBA iPSCs exhibited RPS19 haploinsufficiency, reduction in the 40S/60S ribosomal subunit ratio and early erythroid differentiation arrest in the absence of eltrombopag, compared to control isogenic iPSCs established by CRISPR/Cas9-mediated correction of the RPS19 point mutation. Notably, differentiation of DBA iPSCs in the presence of eltrombopag markedly improved erythroid maturation. Consistent with a molecular mechanism based on intracellular iron chelation, we observed that deferasirox, a clinically licensed iron chelator able to permeate into cells, also enhanced erythropoiesis in our DBA iPSC model. In contrast, erythroid maturation did not improve substantially in DBA iPSC differentiation cultures supplemented with deferoxamine, a clinically available iron chelator that poorly accesses LIP within cellular compartments. These findings identify eltrombopag as a promising new therapeutic to improve anemia in DBA.
Asunto(s)
Anemia de Diamond-Blackfan/tratamiento farmacológico , Anemia de Diamond-Blackfan/patología , Benzoatos/uso terapéutico , Diferenciación Celular , Células Eritroides/patología , Hidrazinas/uso terapéutico , Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Pirazoles/uso terapéutico , Anemia de Diamond-Blackfan/genética , Animales , Secuencia de Bases , Benzoatos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Eritroides/efectos de los fármacos , Eritropoyesis , Humanos , Hidrazinas/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Espacio Intracelular/metabolismo , Hierro/metabolismo , Ratones Endogámicos NOD , Ratones SCID , Mutación/genética , Pirazoles/farmacologíaRESUMEN
Mutations in human equilibrative nucleoside transporter 3 (ENT3) encoded by SLC29A3 results in anemia and erythroid hypoplasia, suggesting that ENT3 may regulate erythropoiesis. Here, we demonstrate that lysosomal ENT3 transport of taurine-conjugated bile acids (TBA) facilitates TBA chemical chaperone function and alleviates endoplasmic reticulum (ER) stress in expanding mouse hematopoietic stem and progenitor cells (HSPCs). Slc29a3-/- HSPCs accumulate less TBA despite elevated levels of TBA in Slc29a3-/- mouse plasma and have elevated basal ER stress, reactive oxygen species (ROS), and radiation-induced apoptosis. Reintroduction of ENT3 allows for increased accumulation of TBA into HSPCs, which results in TBA-mediated alleviation of ER stress and erythroid apoptosis. Transplanting TBA-preconditioned HSPCs expressing ENT3 into Slc29a3-/- mice increase bone marrow repopulation capacity and erythroid pool size and prevent early mortalities. Together, these findings suggest a putative role for a facilitative lysosomal transporter in the bile acid regulation of ER stress in mouse HSPCs which may have implications in erythroid biology, the treatment of anemia observed in ENT3-mutated human genetic disorders, and nucleoside analog drug therapy.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Estrés del Retículo Endoplásmico , Células Madre Hematopoyéticas/metabolismo , Lisosomas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ácidos y Sales Biliares/sangre , Transporte Biológico/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Trasplante de Células Madre Hematopoyéticas , Concentración de Iones de Hidrógeno , Lisosomas/efectos de los fármacos , Metabolómica , Ratones , Proteínas de Transporte de Nucleósidos/metabolismo , Taurina/metabolismo , Ácido Tauroquenodesoxicólico/farmacologíaRESUMEN
The electrorotation (ROT) rates of K562 cells accompanied by erythroid differentiation were estimated to identify the differentiation status by using a novel electrorotation device with a microwell arranged on polynomial electrodes. Successive estimations of individual cells were achieved by sequential manipulations which involve trapping of the cell by positive dielectrophoresis (DEP), rotating by ROT, and removing by negative DEP. The ROT rate increased with the differentiation of K562 cells, because the cytoplasm conductivity would increase with an increase of the concentration of iron ions to produce hemoglobin. The ROT rate could be utilized to estimate the stage of cell differentiation without labeling.
Asunto(s)
Ácido Butírico/farmacología , Técnicas Electroquímicas , Células Eritroides/efectos de los fármacos , Ácido Butírico/química , Diferenciación Celular/efectos de los fármacos , Conductividad Eléctrica , Humanos , Células K562RESUMEN
Hematopoiesis, the complex developmental process that forms blood components and replenishes the blood system, involves multiple intracellular and extracellular mechanisms. We previously demonstrated that lysophosphatidic acid (LPA), a lipid growth factor, has opposing regulatory effects on erythrocyte differentiation through activation of LPA receptors 2 and 3; yet the mechanisms underlying this process remain unclear. In this study, LPA2 is observed that highly expressed in common myeloid progenitors (CMP) in murine myeloid cells, whereas the expression of LPA3 displaces in megakaryocyte-erythroid progenitors (MEP) of later stage of myeloid differentiation. Therefore, we hypothesized that the switching expression of LPA2 and LPA3 determine the hematic homeostasis of mammalian megakaryocytic-erythroid lineage. In vitro colony-forming unit assays of murine progenitors reveal that LPA2 agonist GRI reduces the erythroblast differentiation potential of CMP. In contrast, LPA3 agonist OMPT increases the production of erythrocytes from megakaryocyte-erythrocyte progenitor cells (MEP). In addition, treatment with GRI reduces the erythroid, CMP, and MEP populations in mice, indicating that LPA2 predominantly inhibits myeloid differentiation at an early stage. In contrast, activation of LPA3 increases the production of terminally differentiated erythroid cells through activation of erythropoietic transcriptional factor. We also demonstrate that the LPA3 signaling is essential for restoration of phenylhydrazine (PHZ)-induced acute hemolytic anemia in mice and correlates to erythropoiesis impairment of Hutchinson-Gilford progeria Symptom (HGPS) premature aging expressed K562 model. Our results reveal the distinct roles of LPA2 and LPA3 at different stages of hematopoiesis in vivo, providing potentiated therapeutic strategies of anemia treatment.
Asunto(s)
Anemia Hemolítica/genética , Células Eritroides/metabolismo , Eritropoyesis/genética , Células Mieloides/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Células Madre/metabolismo , Anemia Hemolítica/inducido químicamente , Anemia Hemolítica/tratamiento farmacológico , Anemia Hemolítica/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Modelos Animales de Enfermedad , Células Eritroides/citología , Células Eritroides/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Isoquinolinas/farmacología , Células K562 , Lisofosfolípidos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Células Mieloides/citología , Células Mieloides/efectos de los fármacos , Organotiofosfatos/farmacología , Fenilhidrazinas/administración & dosificación , Ácidos Fosfatidicos/farmacología , Receptores del Ácido Lisofosfatídico/agonistas , Receptores del Ácido Lisofosfatídico/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
BACKGROUND: The use of medicinal agents to augment the fetal hemoglobin (HbF) accretion is an important approach for the treatment of sickle-cell anemia and ß-thalassemia. HbF inducers have the potential to reduce the clinical symptoms and blood transfusion dependence in the patients of ß- hemoglobinopathies. OBJECTIVE: The current study was aimed to examine the erythroid induction potential of newly synthesized thiourea derivatives. METHODS: Thiourea derivatives 1-27 were synthesized by using environmentally friendly methods. Compounds 3, 10 and 22 were found to be new. The structures of synthesized derivatives were deduced by using various spectroscopic techniques. These derivatives were then evaluated for their erythroid induction using the human erythroleukemic K562 cell line, as a model. The benzidine-H2O2 assay was used to evaluate erythroid induction, while HbF expression was studied through immunocytochemistry using the Anti-HbF antibody. Cytotoxicity of compounds 1-27 was also evaluated on mouse fibroblast 3T3 cell line and cancer Hela cell line using MTT assay. RESULT: All the compounds (1-27) have not been reported for their erythroid induction activity previously. Compounds 1, 2, and 3 were found to be the potent erythroid inducing agents with % induction of 45± 6.9, 44± 5.9, and 41± 6.1, at 1.56, 0.78, and 0.78 µM concentrations, respectively, as compared to untreated control (12 ± 1 % induction). Furthermore, compound 1, 2, and 3 significantly induced fetal hemoglobin the expression up to 4.2-fold, 4.06-fold, and 3.52-fold, respectively, as compared to untreated control. Moreover, the compounds 1-4, 6-9, 11, 12, 15, 17, 19, 22, 23, and 25 were found to be non-cytotoxic against the 3T3 cell line. CONCLUSION: This study signifies that the compounds reported here may serve as the starting point for the designing and development of new fetal hemoglobin inducers for the treatment of ß- hemoglobinopathies.
Asunto(s)
Tiourea/análogos & derivados , Tiourea/síntesis química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Células Eritroides/efectos de los fármacos , Tecnología Química Verde , Humanos , Ratones , Estructura Molecular , Tiourea/químicaRESUMEN
The CCDC26 gene is considered to encode a functional noncoding RNA associated with acute myeloid leukemia and other cancers. However, investigations into the physiological roles of CCDC26 are rare. Previously, we reported that CCDC26 regulated proliferation and cell death of leukemia cells through KIT, a receptor tyrosine kinase, by using K562 leukemia cells and their derivative CCDC26-knockdown (KD) cells. Here we propose a new role of CCDC26 in the differentiation of erythroid cells. We showed that expression of embryonic (ε- and ζ-) globins was markedly upregulated in CCDC26-KD cells compared with K562 control cells during hemin-induced differentiation. In contrast, expression of fetal-type γ-globin, a major globin expressed in original K562 cells, was decreased. These changes in the expression of globin genes mainly took place at the transcriptional level, with significant suppression of transcription of adult (ß-, δ-) globins in CCDC26-KD cells. Re-introduction of exogenous CCDC26 into the CCDC26-KD cells recovered low-level expression of the embryonal globins. These results suggest CCDC26 has a role in switching transcription of globin genes in the differentiation of erythroid cells. The expression profile of the CCDC26-KD cells and control cells suggests FOG-2, a transcriptional modulator, as a candidate for a mediator of the CCDC26-associated regulation. We showed that both embryonic globins were transcriptionally activated in FOG-2-KD K562 cells. The KIT inhibitor ISCK03 suppressed the production of hemoglobin in K562 cells but did not affect transcription of globin genes. To summarize, FOG-2, but not KIT, is responsible for globin transcriptional regulation by CCDC26.
Asunto(s)
Proteínas de Unión al ADN/genética , Células Eritroides/citología , Globinas/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN Largo no Codificante/genética , Factores de Transcripción/genética , Diferenciación Celular/efectos de los fármacos , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Regulación del Desarrollo de la Expresión Génica , Globinas/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Imidazoles/farmacología , Células K562 , Sulfonamidas/farmacologíaRESUMEN
While red blood cells (RBCs) and granulocytes have been more studied, platelets and reticulocytes are not commonly used in paroxysmal nocturnal hemoglobinuria (PNH) flow-cytometry and less is known about susceptibility to complement-mediated destruction and effects of anti-complement therapy on these populations. We performed flow-cytometry of RBCs and granulocytes in 90 PNH patients and of platelets and reticulocytes in a subgroup (N = 36), to unveil perturbations of these populations during PNH disease course before and after anti-complement treatment. We found that platelets and reticulocytes were less sensitive to complement-mediated lysis than RBCs but not as resistant as granulocytes, as shown by mean sensitive fraction (difference in a given PNH population vs. PNH granulocyte clone size). In treated patients, reticulocytes, platelets, RBCs (with differences between type II and III) and granulocytes significantly increased post-treatment, confirming the role of PNH hematopoiesis within the context of anti-complement therapy. Moreover, we found that PNH platelet clone size reflects PNH granulocyte clone size. Finally, we established correlations between sensitive fraction of PNH cell-types and thrombosis. In sum, we applied a flow-cytometry panel for investigation of PNH peripheral blood populations' perturbations before and after eculizumab treatment to explore complement-sensitivity and kinetics of these cells during the disease course.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Células Sanguíneas/efectos de los fármacos , Inactivadores del Complemento/uso terapéutico , Hemoglobinuria Paroxística/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/farmacología , Células Sanguíneas/citología , Plaquetas/citología , Plaquetas/efectos de los fármacos , Inactivadores del Complemento/farmacología , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Células Eritroides/citología , Células Eritroides/efectos de los fármacos , Femenino , Citometría de Flujo , Granulocitos/citología , Granulocitos/efectos de los fármacos , Hemoglobinuria Paroxística/sangre , Humanos , Masculino , Persona de Mediana Edad , Reticulocitos/citología , Reticulocitos/efectos de los fármacos , Adulto JovenRESUMEN
Rat developmental toxicity including embryolethality and teratogenicity (mainly ventricular septal defects and wavy ribs) were produced by S-53482, an N-phenylimide herbicide that inhibits protoporphyrinogen oxidase (PPO) common to chlorophyll and heme biosynthesis. The sequence of key biological events in the mode of action has been elucidated as follows: inhibition of PPO interferes with normal heme synthesis, which causes loss of blood cells leading to fetal anemia, embryolethality and the development of malformations. In this study we investigated whether the rat is a relevant model for the assessment of the human hazard of the herbicide. To study effects on heme biosynthesis, human erythroleukemia, human cord blood, and rat erythroleukemia cells were treated with the herbicide during red cell differentiation. Protoporphyrin IX, a marker of PPO inhibition, and heme were determined. We investigated whether synchronous maturation of primitive erythropoiesis, which can contribute to massive losses of embryonic blood, occurs in rats. The population of primitive erythroblasts was observed on gestational days 11 through 14. Heme production was suppressed in rat erythroid cells. In contrast, heme reduction was not seen in both human erythroid cells when PPO was inhibited. Rats underwent synchronous maturation in primitive erythropoiesis. Our results combined with epidemiological findings that patients with deficient PPO are not anemic led us to conclude that human erythroblasts are resistant to the herbicide. It is suggested that the rat would be an inappropriate model for assessing the developmental toxicity of S-53482 in humans as rats are specifically sensitive to PPO inhibition by the herbicide.
Asunto(s)
Benzoxazinas/toxicidad , Células Eritroides/efectos de los fármacos , Hemo/metabolismo , Herbicidas/toxicidad , Ftalimidas/toxicidad , Protoporfirinógeno-Oxidasa/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Células Eritroides/metabolismo , Femenino , Sangre Fetal , Humanos , Embarazo , Protoporfirinas/metabolismo , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
Erythropoietin (EPO) is an essential hormone for erythropoiesis, protecting differentiating erythroblasts against apoptosis. EPO has been largely studied in stress or pathological conditions but its regulatory role in steady state erythropoiesis has been less documented. Herein, we report production of EPO by bone marrow-derived macrophages (BMDM) in vitro, and its further enhancement in BMDM conditioned with media from apoptotic cells. Confocal microscopy confirmed EPO production in erythroblastic island (EBI)-associated macrophages, and analysis of mice depleted of EBI macrophages by clodronate liposomes revealed drops in EPO levels in bone marrow (BM) cell lysates, and decreased percentages of EPO-responsive erythroblasts in the BM. We hypothesize that EBI macrophages are an in-situ source of EPO and sustain basal erythropoiesis in part through its secretion. To study this hypothesis, mice were injected with clodronate liposomes and were supplied with exogenous EPO (1-10 IU/mouse) to evaluate potential rescue of the deficiency in erythroid cells. Our results show that at doses of 5 and 10 IU, EPO significantly rescues BM steady state erythropoiesis in mice deficient of macrophages. We propose existence of a mechanism by which EBI macrophages secrete EPO in response to apoptotic erythroblasts, which is in turn controlled by the numbers of erythroid precursors generated.
Asunto(s)
Apoptosis/efectos de los fármacos , Eritropoyesis/genética , Eritropoyetina/genética , Macrófagos/metabolismo , Animales , Diferenciación Celular/genética , Ácido Clodrónico/farmacología , Eritroblastos/efectos de los fármacos , Eritroblastos/metabolismo , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Eritropoyesis/efectos de los fármacos , Eritropoyetina/farmacología , Liposomas/farmacología , Macrófagos/efectos de los fármacos , RatonesRESUMEN
The hemostimulating effects of c-Jun N-terminal kinase (JNK) inhibitor were examined on the mouse model of myelosuppression provoked by 5-fluorouracil. Blockade of JNK during postcytostatic period accelerated recovery of granulomonocytopoiesis and erythropoiesis. It also increased the content of neutrophilic granulocytes and erythroid cells in the hematopoietic tissue and elevated the counts of neutrophils and reticulocytes in the peripheral blood. The development of these phenomena resulted from elevated content and up-regulated functional activity of bone marrow hematopoietic progenitors associated with the direct action of JNK inhibitor on these progenitors and enhanced secretion of hemopoietins by stromal elements of the hematopoiesis-inducing microenvironment.
Asunto(s)
Citostáticos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Animales , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Fluorouracilo/farmacología , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
FAM122A is a highly conserved housekeeping gene, but its physiological and pathophysiological roles remain greatly elusive. Based on the fact that FAM122A is highly expressed in human CD71+ early erythroid cells, herein we report that FAM122A is downregulated during erythroid differentiation, while its overexpression significantly inhibits erythrocytic differentiation in primary human hematopoietic progenitor cells and erythroleukemia cells. Mechanistically, FAM122A directly interacts with the C-terminal zinc finger domain of GATA1, a critical transcriptional factor for erythropoiesis, and reduces GATA1 chromatin occupancy on the promoters of its target genes, thus resulting in the decrease of GATA1 transcriptional activity. The public datasets show that FAM122A is abnormally upregulated in patients with ß-thalassemia. Collectively, our results demonstrate that FAM122A plays an inhibitory role in the regulation of erythroid differentiation, and it would be a potentially therapeutic target for GATA1-related dyserythropoiesis or an important regulator for amplifying erythroid cells ex vivo.
Asunto(s)
Diferenciación Celular , Células Eritroides/citología , Células Eritroides/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Antígenos CD34/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , ADN/metabolismo , Regulación hacia Abajo/genética , Células Eritroides/efectos de los fármacos , Eritropoyetina/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Células K562 , Fosfoproteínas/química , Unión Proteica/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Dedos de ZincRESUMEN
Congenital erythropoietic porphyria (CEP) is an inborn error of heme synthesis resulting from uroporphyrinogen III synthase (UROS) deficiency and the accumulation of nonphysiological porphyrin isomer I metabolites. Clinical features are heterogeneous among patients with CEP but usually combine skin photosensitivity and chronic hemolytic anemia, the severity of which is related to porphyrin overload. Therapeutic options include symptomatic strategies only and are unsatisfactory. One promising approach to treating CEP is to reduce the erythroid production of porphyrins through substrate reduction therapy by inhibiting 5-aminolevulinate synthase 2 (ALAS2), the first and rate-limiting enzyme in the heme biosynthetic pathway. We efficiently reduced porphyrin accumulation after RNA interference-mediated downregulation of ALAS2 in human erythroid cellular models of CEP disease. Taking advantage of the physiological iron-dependent posttranscriptional regulation of ALAS2, we evaluated whether iron chelation with deferiprone could decrease ALAS2 expression and subsequent porphyrin production in vitro and in vivo in a CEP murine model. Treatment with deferiprone of UROS-deficient erythroid cell lines and peripheral blood CD34+-derived erythroid cultures from a patient with CEP inhibited iron-dependent protein ALAS2 and iron-responsive element-binding protein 2 expression and reduced porphyrin production. Furthermore, porphyrin accumulation progressively decreased in red blood cells and urine, and skin photosensitivity in CEP mice treated with deferiprone (1 or 3 mg/mL in drinking water) for 26 weeks was reversed. Hemolysis and iron overload improved upon iron chelation with full correction of anemia in CEP mice treated at the highest dose of deferiprone. Our findings highlight, in both mouse and human models, the therapeutic potential of iron restriction to modulate the phenotype in CEP.
Asunto(s)
Anemia Hemolítica/tratamiento farmacológico , Deferiprona/uso terapéutico , Quelantes del Hierro/uso terapéutico , Sobrecarga de Hierro/tratamiento farmacológico , Trastornos por Fotosensibilidad/tratamiento farmacológico , Porfiria Eritropoyética/tratamiento farmacológico , 5-Aminolevulinato Sintetasa/antagonistas & inhibidores , 5-Aminolevulinato Sintetasa/biosíntesis , 5-Aminolevulinato Sintetasa/genética , Adulto , Anemia Hemolítica/etiología , Animales , Sistemas CRISPR-Cas , Línea Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Femenino , Técnicas de Sustitución del Gen , Humanos , Hierro/metabolismo , Sobrecarga de Hierro/etiología , Leucemia Eritroblástica Aguda/patología , Ratones , Células Madre de Sangre Periférica/efectos de los fármacos , Células Madre de Sangre Periférica/metabolismo , Trastornos por Fotosensibilidad/etiología , Porfiria Intermitente Aguda/metabolismo , Porfiria Eritropoyética/complicaciones , Porfirinas/biosíntesis , Interferencia de ARN , ARN Interferente Pequeño/farmacologíaRESUMEN
Testosterone stimulates iron-dependent erythropoiesis and suppresses hepcidin. To clarify the role of iron in mediating testosterone's effects on erythropoiesis, we induced iron deficiency in mice by feeding low iron diet. Iron-replete and iron-deficient mice were treated weekly with testosterone propionate or vehicle for 3 weeks. Testosterone treatment increased red cell count in iron-replete mice, but, surprisingly, testosterone reduced red cell count in iron-deficient mice. Splenic stress erythropoiesis was stimulated in iron-deficient mice relative to iron-replete mice, and further increased by testosterone treatment, as indicated by the increase in red pulp area, the number of nucleated erythroblasts, and expression levels of TfR1, GATA1, and other erythroid genes. Testosterone treatment of iron-deficient mice increased the ratio of early-to-late erythroblasts in the spleen and bone marrow, and serum LDH level, consistent with ineffective erythropoiesis. In iron-deficient mice, erythropoietin levels were higher but erythropoietin-regulated genes were generally downregulated relative to iron-replete mice, suggesting erythropoietin resistance. Conclusion: Testosterone treatment stimulates splenic stress erythropoiesis in iron-replete as well as iron-deficient mice. However, testosterone worsens anemia in iron-deficient mice because of ineffective erythropoiesis possibly due to erythropoietin resistance associated with iron deficiency. Iron plays an important role in mediating testosterone's effects on erythropoiesis.