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1.
Blood ; 137(1): 89-102, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-32818241

RESUMEN

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle-arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.


Asunto(s)
Diferenciación Celular/fisiología , Células Eritroides/citología , Eritropoyesis/fisiología , Ribosomas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Células Madre Hematopoyéticas , Humanos , Ratones , Biogénesis de Organelos
2.
Am J Hematol ; 98(12): 1923-1933, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37792521

RESUMEN

Red blood cells (RBC) transfusion is used to alleviate symptoms and prevent complications in anemic patients by restoring oxygen delivery to tissues. RBC transfusion efficacy, that can be measured by a rise in hemoglobin (Hb) concentration, is influenced by donor-, product-, and recipient-related characteristics. In some studies, severe pre-transfusion anemia is associated with a greater than expected Hb increment following transfusion but the biological mechanism underpinning this relationship remains poorly understood. We conducted a prospective study in critically ill patients and quantified Hb increment following one RBC transfusion. In a murine model, we investigated the possibility that, in conjunction with the host erythropoietic response, the persistence of transfused donor RBC is improved to maintain a highest RBC biomass. We confirmed a correlation between a greater Hb increment and a deeper pre-transfusion anemia in a cohort of 17 patients. In the mouse model, Hb increment and post-transfusion recovery were increased in anemic recipients. Post-transfusion RBC recovery was improved in hypoxic mice or those receiving an erythropoiesis-stimulating agent and decreased in those treated with erythropoietin (EPO)-neutralizing antibodies, suggesting that EPO signaling is necessary to observe this effect. Irradiated recipients also showed decreased post-transfusion RBC recovery. The EPO-induced post-transfusion RBC recovery improvement was abrogated in irradiated or in macrophage-depleted recipients, but maintained in splenectomized recipients, suggesting a mechanism requiring erythroid progenitors and macrophages, but which is not spleen-specific. Our study highlights a physiological role of EPO in downregulating post-transfusion RBC clearance, contributing to maintain a vital RBC biomass to rapidly cope with hypoxemia.


Asunto(s)
Anemia , Eritropoyetina , Humanos , Animales , Ratones , Estudios Prospectivos , Anemia/tratamiento farmacológico , Anemia/etiología , Eritropoyetina/farmacología , Eritropoyetina/uso terapéutico , Eritropoyesis/fisiología , Eritrocitos
3.
Blood ; 135(6): 441-448, 2020 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-31826245

RESUMEN

The rare PEL-negative phenotype is one of the last blood groups with an unknown genetic basis. By combining whole-exome sequencing and comparative global proteomic investigations, we found a large deletion in the ABCC4/MRP4 gene encoding an ATP-binding cassette (ABC) transporter in PEL-negative individuals. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Although ABCC4 is an important cyclic nucleotide exporter, red blood cells from ABCC4null/PEL-negative individuals exhibited a normal guanosine 3',5'-cyclic monophosphate level, suggesting a compensatory mechanism by other erythroid ABC transporters. Interestingly, PEL-negative individuals showed an impaired platelet aggregation, confirming a role for ABCC4 in platelet function. Finally, we showed that loss-of-function mutations in the ABCC4 gene, associated with leukemia outcome, altered the expression of the PEL antigen. In addition to ABCC4 genotyping, PEL phenotyping could open a new way toward drug dose adjustment for leukemia treatment.


Asunto(s)
Antígenos de Grupos Sanguíneos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Agregación Plaquetaria , Plaquetas/citología , Plaquetas/metabolismo , Sistemas CRISPR-Cas , Células Eritroides/citología , Células Eritroides/metabolismo , Eliminación de Gen , Humanos , Fenotipo
4.
Haematologica ; 107(2): 403-416, 2022 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-33406814

RESUMEN

APR-246 is a promising new therapeutic agent that targets p53 mutated proteins in myelodysplastic syndromes and in acute myeloid leukemia (AML). APR-246 reactivates the transcriptional activity of p53 mutants by facilitating their binding to DNA target sites. Recent studies in solid cancers have found that APR-246 can also induce p53-independent cell death. In this study, we demonstrate that AML cell death occurring early after APR-246 exposure is suppressed by iron chelators, lipophilic antioxidants and inhibitors of lipid peroxidation, and correlates with the accumulation of markers of lipid peroxidation, thus fulfilling the definition of ferroptosis, a recently described cell death process. The capacity of AML cells to detoxify lipid peroxides by increasing their cystine uptake to maintain major antioxidant molecule glutathione biosynthesis after exposure to APR-246 may be a key determinant of sensitivity to this compound. The association of APR-246 with induction of ferroptosis (either by pharmacological compounds, or genetic inactivation of SLC7A11 or GPX4) had a synergistic effect on the promotion of cell death, both in vivo and ex vivo.


Asunto(s)
Ferroptosis , Leucemia Mieloide Aguda , Muerte Celular , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Quinuclidinas/uso terapéutico
5.
Haematologica ; 107(1): 268-283, 2022 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-33241676

RESUMEN

The gene CXXC5, encoding a Retinoid-Inducible Nuclear Factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome (MDS) and adult acute myeloid leukemia (AML). RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknow. Here, we evaluated the consequences of RINF silencing on cytokineinduced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells (RBCs), without affecting cell viability. The phenotype induced by RINF-silencing was TGFß-dependent and mediated by SMAD7, a TGFß- signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and MDS patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdowndependent erythroid phenotype. Finally, RINF silencing affects 5'-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a Tet2- anchoring platform in mouse. Altogether, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFß superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.


Asunto(s)
Proteínas de Unión al ADN , Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Proteína smad7 , Factores de Transcripción , Adulto , Animales , Ciclo Celular , Epigénesis Genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Síndromes Mielodisplásicos/genética , ARN Mensajero , Proteína smad7/genética
6.
Haematologica ; 106(3): 746-758, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-32327500

RESUMEN

In ribosomopathies, the Diamond-Blackfan anemia (DBA) or 5q- syndrome, ribosomal protein (RP) genes are affected by mutation or deletion, resulting in bone marrow erythroid hypoplasia. Unbalanced production of ribosomal subunits leading to a limited ribosome cellular content regulates translation at the expense of the master erythroid transcription factor GATA1. In RPS14-deficient cells mimicking 5q- syndrome erythroid defects, we show that the transcript length, codon bias of the coding sequence (CDS) and 3'UTR (untranslated region) structure are the key determinants of translation. In these cells, short transcripts with a structured 3'UTR and high codon adaptation index (CAI) showed a decreased translation efficiency. Quantitative analysis of the whole proteome confirmed that the post-transcriptional changes depended on the transcript characteristics that governed the translation efficiency in conditions of low ribosome availability. In addition, proteins involved in normal erythroid differentiation share most determinants of translation selectivity. Our findings thus indicate that impaired erythroid maturation due to 5q- syndrome may proceed from a translational selectivity at the expense of the erythroid differentiation program, and suggest that an interplay between the CDS and UTR may regulate mRNA translation.


Asunto(s)
Anemia de Diamond-Blackfan , Anemia Macrocítica , Proteínas Ribosómicas , Anemia de Diamond-Blackfan/genética , Humanos , Proteoma/genética , Proteínas Ribosómicas/deficiencia , Proteínas Ribosómicas/genética , Ribosomas/genética
7.
Am J Hematol ; 96(4): 480-492, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33476437

RESUMEN

Efficient erythropoiesis relies on the expression of the transferrin receptor type 2 (TFR2). In erythroid precursors, TFR2 facilitates the export of the erythropoietin receptor (EPOR) to cell surface, which ensures the survival and proliferation of erythroblasts. Although TFR2 has a crucial role in erythropoiesis regulation, its mechanism of action remains to be clarified. To understand its role better, we aimed at identifying its protein partners by mass-spectrometry after immunoprecipitation in erythroid cells. Here we report the kinase MRCKα (myotonic dystrophy kinase-related CDC42-binding kinase α) as a new partner of both TFR2 and EPOR in erythroblasts. We show that MRCKα is co-expressed with TFR2, and TFR1 during terminal differentiation and regulates the internalization of the two types of transferrin receptors. The knockdown of MRCKα by shRNA in human primary erythroblasts leads to a decreased cell surface expression of both TFR1 and TFR2, an increased cell-surface expression of EPOR, and a delayed differentiation. Additionally, knockout of Mrckα in the murine MEDEP cells also leads to a striking delay in erythropoiesis, showcasing the importance of this kinase in both species. Our data highlight the importance of MRCKα in the regulation of erythropoiesis.


Asunto(s)
Eritropoyesis/fisiología , Proteína Quinasa de Distrofia Miotónica/fisiología , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Endocitosis , Eritroblastos/citología , Eritroblastos/metabolismo , Técnicas de Inactivación de Genes , Humanos , Hierro/metabolismo , Ratones , Proteína Quinasa de Distrofia Miotónica/aislamiento & purificación , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Receptores de Eritropoyetina/metabolismo , Receptores de Transferrina/metabolismo , Proteína de Unión al GTP cdc42/metabolismo
8.
J Pathol ; 250(3): 251-261, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31729028

RESUMEN

Traditional serrated adenoma (TSA) remains the least understood of all the colorectal adenomas, although these lesions have been associated with a significant cancer risk, twice that of the conventional adenoma (CAD) and of the sessile serrated adenoma (SSA/P). This study was performed to investigate the proteomic profiles of the different colorectal adenomas to better understand the pathogenesis of TSA. We performed a global quantitative proteome analysis using the label-free quantification (LFQ) method on 44 colorectal adenoma (12 TSAs, 15 CADs, and 17 SSA/Ps) and 17 normal colonic mucosa samples, archived as formalin-fixed paraffin-embedded blocks. Unsupervised consensus hierarchical clustering applied to the whole proteomic profile of the 44 colorectal adenomas identified four subtypes: C1 and C2 were well-individualized clusters composed of all the CADs (15/15) and most of the SSA/Ps (13/17), respectively. This is consistent with the fact that CADs and SSA/Ps are homogeneous and distinct colorectal adenoma entities. In contrast, TSAs were subdivided into C3 and C4 clusters, consistent with the more heterogeneous entity of TSA at the morphologic and molecular levels. Comparison of the proteome expression profile between the adenoma subtypes and normal colonic mucosa further confirmed the heterogeneous nature of TSAs, which overlapped either on CADs or SSA/Ps, whereas CADs and SSAs formed homogeneous and distinct entities. Furthermore, we identified LEFTY1 a new potential marker for TSAs that may be relevant for the pathogenesis of TSA. LEFTY1 is an inhibitor of the Nodal/TGFß pathway, which we found to be one of the most overexpressed proteins specifically in TSAs. This finding was confirmed by immunohistochemistry. Our study confirms that CADs and SSA/Ps form homogeneous and distinct colorectal adenoma entities, whereas TSAs are a heterogeneous entity and may arise from either SSA/Ps or from normal mucosa evolving through a process related to the CAD pathway. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Asunto(s)
Adenoma/metabolismo , Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Proteoma , Adenoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Colon/patología , Neoplasias Colorrectales/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Adhesión en Parafina , Proteómica
9.
Nucleic Acids Res ; 47(5): 2681-2698, 2019 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-30726994

RESUMEN

Most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. Here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNA-dependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme, which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eukaryotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems.


Asunto(s)
Núcleo Celular/genética , Citoplasma/genética , ARN Polimerasas Dirigidas por ADN/genética , Transcripción Genética , Animales , Células CHO , Cricetulus , Citoplasma/química , ARN Polimerasas Dirigidas por ADN/química , Células Eucariotas/química , Células Eucariotas/metabolismo , Humanos , Poli A/genética , Poliadenilación/genética
10.
J Cell Sci ; 131(15)2018 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-29976560

RESUMEN

Although the kinase CHK1 is a key player in the DNA damage response (DDR), several studies have recently provided evidence of DDR-independent roles of CHK1, in particular following phosphorylation of its S280 residue. Here, we demonstrate that CHK1 S280 phosphorylation is cell cycle-dependent and peaks during mitosis. We found that this phosphorylation was catalyzed by the kinase PIM2, whose protein expression was also increased during mitosis. Importantly, we identified polo-like kinase 1 (PLK1) as a direct target of CHK1 during mitosis. Genetic or pharmacological inhibition of CHK1 reduced the activating phosphorylation of PLK1 on T210, and recombinant CHK1 was able to phosphorylate T210 of PLK1 in vitro Accordingly, S280-phosphorylated CHK1 and PLK1 exhibited similar specific mitotic localizations, and PLK1 was co-immunoprecipitated with S280-phosphorylated CHK1 from mitotic cell extracts. Moreover, CHK1-mediated phosphorylation of PLK1 was dependent on S280 phosphorylation by PIM2. Inhibition of PIM proteins reduced cell proliferation and mitotic entry, which was rescued by expressing a T210D phosphomimetic mutant of PLK1. Altogether, these data identify a new PIM-CHK1-PLK1 phosphorylation cascade that regulates different mitotic steps independently of the CHK1 DDR function.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Mitosis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Daño del ADN/genética , Daño del ADN/fisiología , Células HeLa , Humanos , Ratones Noqueados , Mitosis/genética , Fosforilación/genética , Fosforilación/fisiología , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Espectrometría de Masas en Tándem , Quinasa Tipo Polo 1
11.
Haematologica ; 105(9): 2240-2249, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-33054049

RESUMEN

ß-thalassemia major (ß-TM) is an inherited hemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of hemoglobin, leading to the accumulation of free a-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human ß-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free a-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of ß-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of ß-TM.


Asunto(s)
Carioferinas , Receptores Citoplasmáticos y Nucleares , Talasemia beta , Diferenciación Celular , Eritroblastos , Eritropoyesis , Humanos , Carioferinas/genética , Receptores Citoplasmáticos y Nucleares/genética , Talasemia beta/tratamiento farmacológico , Talasemia beta/genética , Proteína Exportina 1
12.
Eur J Haematol ; 105(5): 588-596, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32659848

RESUMEN

OBJECTIVES: Venetoclax combined with hypomethylating agents is a new therapeutic strategy frequently used for treating AML patients who are not eligible for conventional chemotherapy. However, high response rates are heterogeneous due to different mechanisms mediating resistance to venetoclax such as up-regulation of MCL-1 expression. We thus tested the anti-leukemic activity of S63845, a specific MCL-1 inhibitor. METHODS: Apoptosis induces by S63845 with or without venetoclax was evaluated in primary AML samples and in AML cell lines co-cultured or not with bone marrow (BM) mesenchymal stromal cells. Sensitivity of leukemic cells to S63845 was correlated to the expression level of BCL-2, MCL-1, and BCL-XL determined by Western Blot and mass spectrometry-based proteomics. RESULTS: We observed that even if MCL-1 expression is weak compared to BCL-2, S63845 induces apoptosis of AML cells and strongly synergizes with venetoclax. Furthermore, AML cells resistant to venetoclax are highly sensitive to S63845. Interestingly, the synergistic effect of S63845 toward venetoclax-mediated apoptosis of AML cells is still observed in a context of interaction with the BM microenvironment that intrinsically mediates resistance to BCL2 inhibition. CONCLUSION: These results are therefore of great relevance for clinicians as they provide the rational for combining BCL-2 and MCL-1 inhibition in AML.


Asunto(s)
Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Pirimidinas/farmacología , Sulfonamidas/farmacología , Tiofenos/farmacología , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirimidinas/administración & dosificación , Sulfonamidas/administración & dosificación , Tiofenos/administración & dosificación
13.
Haematologica ; 104(5): 907-918, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30309849

RESUMEN

AMP-activated protein kinase (AMPK) is a heterotrimeric complex containing α, ß, and γ subunits involved in maintaining integrity and survival of murine red blood cells. Indeed, Ampk α1-/- , Ampk ß1-/- and Ampk γ1-/- mice develop hemolytic anemia and the plasma membrane of their red blood cells shows elasticity defects. The membrane composition evolves continuously along erythropoiesis and during red blood cell maturation; defects due to the absence of Ampk could be initiated during erythropoiesis. We, therefore, studied the role of AMPK during human erythropoiesis. Our data show that AMPK activation had two distinct phases in primary erythroblasts. The phosphorylation of AMPK (Thr172) and its target acetyl CoA carboxylase (Ser79) was elevated in immature erythroblasts (glycophorin Alow), then decreased conjointly with erythroid differentiation. In erythroblasts, knockdown of the α1 catalytic subunit by short hairpin RNA led to a decrease in cell proliferation and alterations in the expression of membrane proteins (band 3 and glycophorin A) associated with an increase in phosphorylation of adducin (Ser726). AMPK activation in mature erythroblasts (glycophorin Ahigh), achieved through the use of direct activators (GSK621 and compound 991), induced cell cycle arrest in the S phase, the induction of autophagy and caspase-dependent apoptosis, whereas no such effects were observed in similarly treated immature erythroblasts. Thus, our work suggests that AMPK activation during the final stages of erythropoiesis is deleterious. As the use of direct AMPK activators is being considered as a treatment in several pathologies (diabetes, acute myeloid leukemia), this observation is pivotal. Our data highlighted the importance of the finely-tuned regulation of AMPK during human erythropoiesis.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Diferenciación Celular , Eritroblastos/citología , Eritropoyesis , Regulación Enzimológica de la Expresión Génica , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/genética , Adulto , Animales , Apoptosis , Autofagia , Células Cultivadas , Activación Enzimática , Eritroblastos/metabolismo , Humanos , Ratones , Ratones Noqueados , Fosforilación , ARN Interferente Pequeño/genética
14.
Haematologica ; 103(6): 972-981, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29599206

RESUMEN

Polycythemia vera is a chronic myeloproliferative neoplasm characterized by the JAK2V617F mutation, elevated blood cell counts and a high risk of thrombosis. Although the red cell lineage is primarily affected by JAK2V617F, the impact of mutated JAK2 on circulating red blood cells is poorly documented. Recently, we showed that in polycythemia vera, erythrocytes had abnormal expression of several proteins including Lu/BCAM adhesion molecule and proteins from the endoplasmic reticulum, mainly calreticulin and calnexin. Here we investigated the effects of hydroxycarbamide and interferon-α treatments on the expression of erythroid membrane proteins in a cohort of 53 patients. Surprisingly, while both drugs tended to normalize calreticulin expression, proteomics analysis showed that hydroxycarbamide deregulated the expression of 53 proteins in red cell ghosts, with overexpression and downregulation of 37 and 16 proteins, respectively. Within over-expressed proteins, hydroxycarbamide was found to enhance the expression of adhesion molecules such as Lu/BCAM and CD147, while interferon-α did not. In addition, we found that hydroxycarbamide increased Lu/BCAM phosphorylation and exacerbated red cell adhesion to its ligand laminin. Our study reveals unexpected adverse effects of hydroxycarbamide on red cell physiology in polycythemia vera and provides new insights into the effects of this molecule on gene regulation and protein recycling or maturation during erythroid differentiation. Furthermore, our study shows deregulation of Lu/BCAM and CD147 that are two ubiquitously expressed proteins linked to progression of solid tumors, paving the way for future studies to address the role of hydroxycarbamide in tissues other than blood cells in myeloproliferative neoplasms.


Asunto(s)
Moléculas de Adhesión Celular/genética , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hidroxiurea/farmacología , Proteínas de la Membrana/genética , Policitemia Vera/genética , Alelos , Biomarcadores , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/patología , Femenino , Humanos , Janus Quinasa 2/genética , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Mutación , Policitemia Vera/sangre , Policitemia Vera/diagnóstico
15.
Blood ; 126(11): 1346-56, 2015 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-26186940

RESUMEN

Cancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34(+) progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC(K320A) allele and the addition of the tricarboxyclic acid cycle product α-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML.


Asunto(s)
Glutamina/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Bencenoacetamidas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclo del Ácido Cítrico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Glutaminasa/antagonistas & inhibidores , Glutaminasa/genética , Glutaminasa/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Mitocondrias/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Sulfonamidas/farmacología , Tiadiazoles/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
16.
Curr Opin Hematol ; 22(3): 193-8, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25767952

RESUMEN

PURPOSE OF REVIEW: The type 1 transferrin receptor (TfR1) is well known as a key player in erythroid differentiation through its role in iron uptake. Recently, it has been demonstrated that TfR1 could also have signaling functions in erythroid cells. Moreover, the second transferrin receptor, TfR2, whose signaling functions in hepatic cells are well established, was recently shown to be a partner of the erythropoietin receptor (EpoR) and thereby likely to play a role in erythroid differentiation. RECENT FINDINGS: This review reports recent findings regarding the specificities of the regulation of TfR1 expression and iron uptake in erythroblasts. The newly discovered noncanonical actions of TfR1 and TfR2 in erythroid cells are also discussed. SUMMARY: Erythrocytes contain more than 60% of the iron of the body and each day, differentiating erythroid cells uptake around 20 mg of iron for heme synthesis. Accordingly, TfR1 is one of the most abundant membrane proteins of the erythroblasts and it is not surprising that specific regulations regarding both its expression and its mechanism of action operate in erythroblasts. The signaling functions of both TfR1 and TfR2 in erythroid cells were unexpected and these recent findings open a new field of research regarding the last steps of erythroid differentiation and their regulation.


Asunto(s)
Eritropoyesis/fisiología , Receptores de Transferrina/metabolismo , Diferenciación Celular , Humanos , Hierro/metabolismo
17.
Blood ; 122(20): 3521-32, 2013 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-24014241

RESUMEN

Cancer cells require nutrients and energy to adapt to increased biosynthetic activity, and protein synthesis inhibition downstream of mammalian target of rapamycin complex 1 (mTORC1) has shown promise as a possible therapy for acute myeloid leukemia (AML). Glutamine contributes to leucine import into cells, which controls the amino acid/Rag/mTORC1 signaling pathway. We show in our current study that glutamine removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of the SLC1A5 high-affinity transporter for glutamine induces apoptosis and inhibits tumor formation in a mouse AML xenotransplantation model. l-asparaginase (l-ase) is an anticancer agent also harboring glutaminase activity. We show that l-ases from both Escherichia coli and Erwinia chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that this inhibition correlates with their glutaminase activity levels and produces a strong apoptotic response in primary AML cells. We further show that l-ases upregulate glutamine synthase (GS) expression in leukemic cells and that a GS knockdown enhances l-ase-induced apoptosis in some AML cells. Finally, we observe a strong autophagic process upon l-ase treatment. These results suggest that l-ase anticancer activity and glutamine uptake inhibition are promising new therapeutic strategies for AML.


Asunto(s)
Glutamina/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Sistema de Transporte de Aminoácidos ASC/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos ASC/genética , Animales , Apoptosis/efectos de los fármacos , Asparaginasa/aislamiento & purificación , Asparaginasa/farmacología , Autofagia/efectos de los fármacos , Proteínas Bacterianas/farmacología , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Dickeya chrysanthemi/enzimología , Ensayos de Selección de Medicamentos Antitumorales , Proteínas de Escherichia coli/farmacología , Femenino , Glutaminasa/aislamiento & purificación , Glutaminasa/farmacología , Glutamina/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Leucemia Mielomonocítica Aguda/tratamiento farmacológico , Leucemia Mielomonocítica Aguda/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Desnudos , Persona de Mediana Edad , Antígenos de Histocompatibilidad Menor , Complejos Multiproteicos/antagonistas & inhibidores , Biosíntesis de Proteínas/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto Joven
18.
Haematologica ; 100(4): 458-65, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25637053

RESUMEN

Transferrin receptor-2 is a transmembrane protein whose expression is restricted to hepatocytes and erythroid cells. Transferrin receptor-2 has a regulatory function in iron homeostasis, since its inactivation causes systemic iron overload. Hepatic transferrin receptor-2 participates in iron sensing and is involved in hepcidin activation, although the mechanism remains unclear. Erythroid transferrin receptor-2 associates with and stabilizes erythropoietin receptors on the erythroblast surface and is essential to control erythrocyte production in iron deficiency. We identified a soluble form of transferrin receptor-2 in the media of transfected cells and showed that cultured human erythroid cells release an endogenous soluble form. Soluble transferrin receptor-2 originates from a cleavage of the cell surface protein, which is inhibited by diferric transferrin in a dose-dependent manner. Accordingly, the shedding of the transferrin receptor-2 variant G679A, mutated in the Arginine-Glycine-Aspartic acid motif and unable to bind diferric transferrin, is not modulated by the ligand. This observation links the process of transferrin receptor-2 removal from the plasma membrane to iron homeostasis. Soluble transferrin receptor-2 does not affect the binding of erythropoietin to erythropoietin receptor or the consequent signaling and partially inhibits hepcidin promoter activation only in vitro. Whether it is a component of the signals released by erythropoiesis in iron deficiency remains to be investigated. Our results indicate that membrane transferrin receptor-2, a sensor of circulating iron, is released from the cell membrane in iron deficiency.


Asunto(s)
Membrana Celular/metabolismo , Hierro/metabolismo , Receptores de Transferrina/metabolismo , Antígenos CD/metabolismo , Línea Celular , Células Eritroides/metabolismo , Eritropoyetina/metabolismo , Expresión Génica , Hepcidinas/genética , Humanos , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Transporte de Proteínas , Proteolisis , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Receptores de Transferrina/sangre , Receptores de Transferrina/genética , Activación Transcripcional , Transferrina/metabolismo
20.
Blood ; 119(18): 4228-41, 2012 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-22438255

RESUMEN

Oncogenic mutations leading to persistent kinase activities are associated with malignancies. Therefore, deciphering the signaling networks downstream of these oncogenic stimuli remains a challenge to gather insights into targeted therapy. To elucidate the biochemical networks connecting the Kit mutant to leukemogenesis, in the present study, we performed a global profiling of tyrosine-phosphorylated proteins from mutant Kit-driven murine leukemia proerythroblasts and identified Shp2 and Stat5 as proximal effectors of Kit. Shp2 or Stat5 gene depletion by sh-RNA, combined with pharmacologic inhibition of PI3kinase or Mek/Erk activities, revealed 2 distinct and independent signaling pathways contributing to malignancy. We demonstrate that cell survival is driven by the Kit/Shp2/Ras/Mek/Erk1/2 pathway, whereas the G(1)/S transition during the cell cycle is accelerated by both the Kit/Stat5 and Kit/PI3K/Akt pathways. The combined use of the clinically relevant drugs NVP-BEZ235, which targets the cell cycle, and Obatoclax, which targets survival, demonstrated synergistic effects to inhibit leukemia cell growth. This synergy was confirmed with a human mast leukemia cell line (HMC-1.2) that expresses mutant Kit. The results of the present study using liquid chromatography/tandem mass spectrometry analysis have elucidated signaling networks downstream of an oncogenic kinase, providing a molecular rationale for pathway-targeted therapy to treat cancer cells refractory to tyrosine kinase inhibitors.


Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Adenilato Quinasa/antagonistas & inhibidores , Adenilato Quinasa/fisiología , Animales , Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Imidazoles/farmacología , Indoles , Leucemia de Mastocitos/patología , Ratones , Ratones Desnudos , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Fosfotirosina/análisis , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/fisiología , Pirroles/farmacología , Quinolinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/fisiología , Ensayo de Tumor de Célula Madre
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