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Coagulation disorders are described in COVID-19 and long COVID patients. In particular, SARS-CoV-2 infection in megakaryocytes, which are precursors of platelets involved in thrombotic events in COVID-19, long COVID and, in rare cases, in vaccinated individuals, requires further investigation, particularly with the emergence of new SARS-CoV-2 variants. CD147, involved in the regulation of inflammation and required to fight virus infection, can facilitate SARS-CoV-2 entry into megakaryocytes. MCT4, a co-binding protein of CD147 and a key player in the glycolytic metabolism, could also play a role in SARS-CoV-2 infection. Here, we investigated the susceptibility of megakaryocytes to SARS-CoV-2 infection via CD147 and MCT4. We performed infection of Dami cells and human CD34+ hematopoietic progenitor cells induced to megakaryocytic differentiation with SARS-CoV-2 pseudovirus in the presence of AC-73 and syrosingopine, respective inhibitors of CD147 and MCT4 and inducers of autophagy, a process essential in megakaryocyte differentiation. Both AC-73 and syrosingopine enhance autophagy during differentiation but only AC-73 enhances megakaryocytic maturation. Importantly, we found that AC-73 or syrosingopine significantly inhibits SARS-CoV-2 infection of megakaryocytes. Altogether, our data indicate AC-73 and syrosingopine as inhibitors of SARS-CoV-2 infection via CD147 and MCT4 that can be used to prevent SARS-CoV-2 binding and entry into megakaryocytes, which are precursors of platelets involved in COVID-19-associated coagulopathy.
Assuntos
Megacariócitos , Fenóis , Reserpina , SARS-CoV-2 , Humanos , COVID-19 , Megacariócitos/virologia , Fenóis/farmacologia , Síndrome de COVID-19 Pós-Aguda , Reserpina/análogos & derivados , Reserpina/farmacologia , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacosRESUMO
Autophagy is a highly conserved cellular degradation process that regulates cellular metabolism and homeostasis under normal and pathophysiological conditions. Autophagy and metabolism are linked in the hematopoietic system, playing a fundamental role in the self-renewal, survival, and differentiation of hematopoietic stem and progenitor cells, and in cell death, particularly affecting the cellular fate of the hematopoietic stem cell pool. In leukemia, autophagy sustains leukemic cell growth, contributes to survival of leukemic stem cells and chemotherapy resistance. The high frequency of disease relapse caused by relapse-initiating leukemic cells resistant to therapy occurs in acute myeloid leukemia (AML), and depends on the AML subtypes and treatments used. Targeting autophagy may represent a promising strategy to overcome therapeutic resistance in AML, for which prognosis remains poor. In this review, we illustrate the role of autophagy and the impact of its deregulation on the metabolism of normal and leukemic hematopoietic cells. We report updates on the contribution of autophagy to AML development and relapse, and the latest evidence indicating autophagy-related genes as potential prognostic predictors and drivers of AML. We review the recent advances in autophagy manipulation, combined with various anti-leukemia therapies, for an effective autophagy-targeted therapy for AML.
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Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular , Autofagia , BiologiaRESUMO
BACKGROUND AND AIMS: Intestinal fibrosis is a common complication of inflammatory bowel diseases. Medical treatment of intestinal fibrosis is an unmet therapeutic need. CD147 overexpression can induce myofibroblast differentiation associated with extracellular matrix deposition, favouring the development of fibrosis. To understand whether CD147 may promote intestinal fibrosis, we analysed its expression and blocked its function by using its specific inhibitor AC-73 [3-{2-[([1,1'-biphenyl]-4-ylmethyl) amino]-1-hydroxyethyl} phenol] in the murine TNBS [trinitrobenzenesulfonic acid]-chronic colitis model associated with intestinal fibrosis. METHODS: TNBS chronic colitis was induced by weekly intrarectal administration of escalating doses of TNBS. Ethanol-treated and untreated mice were used as controls. Separated groups of TNBS, ethanol-treated or untreated mice received AC-73 or vehicle administered intraperitoneally from day 21 to day 49. At day 49, mice were killed, and colons collected for histological analysis, protein and RNA extraction. CD147, α-SMA and activated TGF-ß1 protein levels, CD147/ERK/STAT3 signalling pathway and autophagy were assessed by Western blot, collagen and inflammatory/fibrogenic cytokines mRNA tissue content by quantitative PCR. RESULTS: In mice with chronic TNBS colitis, CD147 protein level increased during fibrosis development in colonic tissue, as compared to control mice. CD147 inhibition by AC-73 treatment reduced intestinal fibrosis, collagen and cytokine mRNA tissue content, without significant modulation of activated TGF-ß1 protein tissue content. AC-73 inhibited CD147/ERK1/2 and STAT3 signalling pathway activation and induced autophagy. CONCLUSIONS: CD147 is a potential new target for controlling intestinal fibrosis and its inhibitor, AC-73, might represent a potential new anti-fibrotic therapeutic option in IBD.
Assuntos
Basigina , Colite , Fenóis , Fator de Crescimento Transformador beta1 , Animais , Camundongos , Autofagia , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Colágeno/metabolismo , Colo/patologia , Modelos Animais de Doenças , Etanol , Fibrose , Fenóis/farmacologia , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Ácido Trinitrobenzenossulfônico/toxicidade , Basigina/antagonistas & inibidoresRESUMO
Metabolism in acute myeloid leukemia (AML) cells is dependent primarily on oxidative phosphorylation. However, in order to sustain their high proliferation rate and metabolic demand, leukemic blasts use a number of metabolic strategies, including glycolytic metabolism. Understanding whether monocarboxylate transporters MCT1 and MCT4, which remove the excess of lactate produced by cancer cells, represent new hematological targets, and whether their respective inhibitors, AR-C155858 and syrosingopine, can be useful in leukemia therapy, may reveal a novel treatment strategy for patients with AML. We analyzed MCT1 and MCT4 expression and function in hematopoietic progenitor cells from healthy cord blood, in several leukemic cell lines and in primary leukemic blasts from patients with AML, and investigated the effects of AR-C155858 and syrosingopine, used alone or in combination with arabinosylcytosine, on leukemic cell proliferation. We found an inverse correlation between MCT1 and MCT4 expression levels in leukemic cells, and showed that MCT4 overexpression is associated with poor prognosis in AML patients. We also found that AR-C155858 and syrosingopine inhibit leukemic cell proliferation by activating two different cell-death related pathways, i.e., necrosis for AR-C155858 treatment and autophagy for syrosingopine, and showed that AR-C155858 and syrosingopine exert an anti-proliferative effect, additive to chemotherapy, by enhancing leukemic cells sensitivity to chemotherapeutic agents. Altogether, our study shows that inhibition of MCT1 or MCT4 impairs leukemic cell proliferation, suggesting that targeting lactate metabolism may be a new therapeutic strategy for AML, and points to MCT4 as a potential therapeutic target in AML patients and to syrosingopine as a new anti-proliferative drug and inducer of autophagy to be used in combination with conventional chemotherapeutic agents in AML treatment.
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CD147 is a transmembrane glycoprotein with multiple functions in human healthy tissues and diseases, in particular in cancer. Overexpression of CD147 correlates with biological functions that promote tumor progression and confers resistance to chemotherapeutic drugs. In contrast to solid tumors, the role of CD147 has not been extensively studied in leukemia. Understanding whether CD147 represents a new hematologic target and whether its inhibitor AC-73 may be used in leukemia therapy may reveal an alternative treatment strategy in patients with acute myeloid leukemia (AML). We analyzed CD147 expression and function in hematopoietic progenitor cells from normal cord blood, in several leukemic cell lines and in primary leukemic blasts obtained from patients with AML. We investigated the effects of AC-73, used alone or in combination with arabinosylcytosine (Ara-C) and arsenic trioxide (ATO), on leukemic cell proliferation. We demonstrated that CD147 overexpression promotes leukemic cell proliferation. We showed that AC-73 exhibits a potent growth inhibitory activity in leukemic cells, by inhibiting the ERK/STAT3 activation pathway and activating autophagy. We demonstrated that AC-73 exerts an anti-proliferative effect additive to chemotherapy by enhancing leukemic cell sensitivity to Ara-C-induced cytotoxicity or to ATO-induced autophagy. We also reported CD147 expression in the fraction of leukemic blasts expressing CD371, a marker of leukemic stem cells. Altogether, our study indicates CD147 as a novel potential target in the treatment of AML and AC-73 as an anti-proliferative drug and an inducer of autophagy in leukemic cells to use in combination with chemotherapeutic agents.
Assuntos
Antineoplásicos/farmacologia , Autofagia , Basigina/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glicoproteínas/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Fenóis/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Trióxido de Arsênio/farmacologia , Citarabina/farmacologia , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Prognóstico , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
MicroRNAs (miRNAs or miRs) are a class of evolutionarily-conserved small, regulatory non-coding RNAs, 19-3 nucleotides in length, that negatively regulate protein coding gene transcripts' expression. miR-146 (146a and 146b) and miR-155 are among the first and most studied miRs for their multiple roles in the control of the innate and adaptive immune processes and for their deregulation and oncogenic role in some tumors. In the present review, we have focused on the recent acquisitions about the key role played by miR-146a, miR-146b and miR-155 in the control of the immune system and in myeloid tumorigenesis. Growing experimental evidence indicates an opposite role of miR-146a with respect to miR-155 in the fine regulation of many steps of the immune response, acting at the level of the various cell types involved in innate and adaptive immune mechanisms. The demonstration that miR-155 overexpression plays a key pathogenic role in some lymphomas and acute myeloid leukemias has led to the development of an antagomir-based approach as a new promising therapeutic strategy.
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Duchenne Muscular Dystrophy, a genetic disorder that results in a gradual breakdown of muscle, is associated to mild to severe cognitive impairment in about one-third of dystrophic patients. The brain dysfunction is independent of the muscular pathology, occurs early, and is most likely due to defects in the assembly of the Dystrophin-associated Protein Complex (DPC) during embryogenesis. We have recently described the interaction of the DPC component ß-dystrobrevin with members of complexes that regulate chromatin dynamics, and suggested that ß-dystrobrevin may play a role in the initiation of neuronal differentiation. Since oxygen concentrations and miRNAs appear as well to be involved in the cellular processes related to neuronal development, we have studied how these factors act on ß-dystrobrevin and investigated the possibility of their functional interplay using the NTera-2 cell line, a well-established model for studying neurogenesis. We followed the pattern of expression and regulation of ß-dystrobrevin during the early stages of neuronal differentiation induced by exposure to retinoic acid (RA) under hypoxia as compared with normoxia, and found that ß-dystrobrevin expression is regulated during RA-induced differentiation of NTera-2 cells. We also found that ß-dystrobrevin pattern is delayed under hypoxic conditions, together with a delay in the differentiation and an increase in the proliferation rate of cells. We identified miRNA-143 as a direct regulator of ß-dystrobrevin expression, demonstrated that ß-dystrobrevin is expressed in the nucleus and showed that, in line with our previous in vitro results, ß-dystrobrevin is a repressor of synapsin I in live cells. Altogether the newly identified regulatory pathway miR-143/ß-dystrobrevin/synapsin I provides novel insights into the functions of ß-dystrobrevin and opens up new perspectives for elucidating the molecular mechanisms underlying the neuronal involvement in muscular dystrophy.
Assuntos
Proteínas Associadas à Distrofina/genética , Proteínas Associadas à Distrofina/metabolismo , MicroRNAs/genética , Neurogênese , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Regiões 3' não Traduzidas , Diferenciação Celular , Hipóxia Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Proliferação de Células , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
The main hematopoietic stem cell (HSC) functions, self-renewal and differentiation, are finely regulated by both intrinsic mechanisms such as transcriptional and epigenetic regulators and extrinsic signals originating in the bone marrow microenvironment (HSC niche) or in the body (humoral mediators). The interaction between regulatory signals and cellular metabolism is an emerging area. Several metabolic pathways function differently in HSCs compared with progenitors and differentiated cells. Hypoxia, acting through hypoxia-inducing factors, has emerged as a key regulator of stem cell biology and acts by maintaining HSC quiescence and a condition of metabolic dormancy based on anaerobic glycolytic energetic metabolism, with consequent low production reactive oxygen species (ROS) and high antioxidant defense. Hematopoietic cell differentiation is accompanied by changes in oxidative metabolism (decrease of anaerobic glycolysis and increase of oxidative phosphorylation) and increased levels of ROS. Leukemic stem cells, defined as the cells that initiate and maintain the leukemic process, show peculiar metabolic properties in that they are more dependent on oxidative respiration than on glycolysis and are more sensitive to oxidative stress than normal HSCs. Several mitochondrial abnormalities have been described in acute myeloid leukemia (AML) cells, explaining the shift to aerobic glycolysis observed in these cells and offering the unique opportunity for therapeutic metabolic targeting. Finally, frequent mutations of the mitochondrial isocitrate dehydrogenase-2 (IDH2) enzyme are observed in AML cells, in which the mutated enzyme acts as an oncogenic driver and can be targeted using specific inhibitors under clinical evaluation with promising results.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Hipóxia/metabolismo , Leucemia/metabolismo , Células-Tronco Neoplásicas/metabolismo , Estresse Oxidativo , Animais , Diferenciação Celular , Metabolismo Energético , Glutationa/metabolismo , Glicólise , Células-Tronco Hematopoéticas/citologia , Humanos , Leucemia/genética , Mitocôndrias/metabolismo , Mutação , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
High expression of the chemokine receptor 4, CXCR4, associated with a negative prognosis in acute myeloid leukemia, is related to hypoxia. Because CXCR4 expression is under the post-transcriptional control of microRNA-146a in normal and leukemic monocytic cells, we first investigated the impact of hypoxia on microRNA-146a and CXCR4 expression during monocytopoiesis and in acute monocytic leukemia. We then analyzed the effects of hypoxia on drug sensitivity of CXCR4-expressing leukemic cells. We found that microRNA-146a is a target of hypoxia-inducible factor-1α or -2α in relation to the stage of monocytopoiesis and the level of hypoxia, and demonstrated the regulation of the microRNA-146a/CXCR4 pathway by hypoxia in monocytes derived from CD34(+) cells. Thus, in myeloid leukemic cell lines, hypoxia-mediated control of the microRNA-146a/CXCR4 pathway depends only on the capacity of hypoxia-inducible factor-1α to up-regulate microRNA-146a, which in turn decreases CXCR4 expression. However, at variance with normal monocytic cells and leukemic cell lines, in acute monocytic leukemia overexpressing CXCR4, hypoxia up-modulates microRNA-146a but fails to down-modulate CXCR4 expression. We then investigated the effect of hypoxia on the response of leukemic cells to chemotherapy alone or in combination with stromal-derived factor-1α. We found that hypoxia increases stromal-derived factor-1α-induced survival of leukemic cells by decreasing their sensitivity to anti-leukemic drugs. Altogether, our results demonstrate that hypoxia-mediated regulation of microRNA-146a, which controls CXCR4 expression in monocytic cells, is lost in acute monocytic leukemia, thus contributing to maintaining CXCR4 overexpression and protecting the cells from anti-leukemic drugs in the hypoxic bone marrow microenvironment.
Assuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Monocítica Aguda , MicroRNAs/biossíntese , Monócitos/metabolismo , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/biossíntese , Receptores CXCR4/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular/efeitos dos fármacos , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leucemia Monocítica Aguda/tratamento farmacológico , Leucemia Monocítica Aguda/metabolismo , Leucemia Monocítica Aguda/patologia , Masculino , Monócitos/patologia , Células U937RESUMO
BACKGROUND: The transmembrane 9 superfamily protein member 4, TM9SF4, belongs to the TM9SF family of proteins highly conserved through evolution. TM9SF4 homologs, previously identified in many different species, were mainly involved in cellular adhesion, innate immunity and phagocytosis. In human, the function and biological significance of TM9SF4 are currently under investigation. However, TM9SF4 was found overexpressed in human metastatic melanoma and in a small subset of acute myeloid leukemia (AMLs) and myelodysplastic syndromes, consistent with an oncogenic function of this gene. PURPOSE AND RESULTS: In this study, we first analyzed the expression and regulation of TM9SF4 in normal and leukemic cells and identified TM9SF4 as a gene highly expressed in human quiescent CD34+ hematopoietic progenitor cells (HPCs), regulated during monocytic and granulocytic differentiation of HPCs, both lineages giving rise to mature myeloid cells involved in adhesion, phagocytosis and immunity. Then, we found that TM9SF4 is markedly overexpressed in leukemic cells and in AMLs, particularly in M2, M3 and M4 AMLs (i.e., in AMLs characterized by the presence of a more or less differentiated granulocytic progeny), as compared to normal CD34+ HPCs. Proliferation and differentiation of HPCs occurs in hypoxia, a physiological condition in bone marrow, but also a crucial component of cancer microenvironment. Here, we investigated the impact of hypoxia on TM9SF4 expression in leukemic cells and identified TM9SF4 as a direct target of HIF-1α, downregulated in these cells by hypoxia. Then, we found that the hypoxia-mediated downregulation of TM9SF4 expression is associated with a decrease of cell adhesion of leukemic cells to fibronectin, thus demonstrating that human TM9SF4 is a new molecule involved in leukemic cell adhesion. CONCLUSIONS: Altogether, our study reports for the first time the expression of TM9SF4 at the level of normal and leukemic hematopoietic cells and its marked expression at the level of AMLs displaying granulocytic differentiation.
Assuntos
Regulação Leucêmica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Leucemia Mieloide Aguda/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Oxigênio/farmacologia , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Fibronectinas/metabolismo , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Granulócitos/patologia , Células HEK293 , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas de Membrana/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/patologia , Proteínas de Neoplasias/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Transdução de Sinais , Células Tumorais CultivadasRESUMO
MicroRNA miR-146a and PLZF are reported as major players in the control of hematopoiesis, immune function and cancer. PLZF is described as a miR-146a repressor, whereas CXCR4 and TRAF6 were identified as miR-146a direct targets in different cell types. CXCR4 is a co-receptor of CD4 molecule that facilitates HIV-1 entry into T lymphocytes and myeloid cells, whereas TRAF6 is involved in immune response. Thus, the role of miR-146a in HIV-1 infection is currently being thoroughly investigated. In this study, we found that PLZF mediates suppression of miR-146a to control increases of CXCR4 and TRAF6 protein levels in human primary CD4(+) T lymphocytes. We show that miR-146a upregulation by AMD3100 treatment or PLZF silencing, decreases CXCR4 protein expression and prevents HIV-1 infection of leukemic monocytic cell line and CD4(+) T lymphocytes. Our findings improve the prospects of developing new therapeutic strategies to prevent HIV-1 entry via CXCR4 by using the PLZF/miR-146a axis.
Assuntos
Linfócitos T CD4-Positivos/virologia , Regulação da Expressão Gênica , HIV-1/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , MicroRNAs/metabolismo , Receptores CXCR4/biossíntese , Receptores de HIV/biossíntese , Adulto , Humanos , Proteína com Dedos de Zinco da Leucemia Promielocítica , Internalização do VírusRESUMO
In addition to contrast human immunodeficiency virus (HIV) replication, the HIV protease inhibitors (HIV-PI) have reduced tumour incidence or clinical progression in infected patients. In this regard, we have previously shown that, independently of its anti-viral activity, the HIV-PI indinavir (IDV) directly blocks matrix metalloproteinase (MMP)-2 proteolytic activation, thus efficiently inhibiting tumour angiogenesis in vitro, in animal models, and in humans. Herein we investigated the molecular mechanism for IDV anti-angiogenic effect. We found that treatment of human primary endothelial cells with therapeutic IDV concentrations decreases the expression of membrane type (MT)1-MMP, which is the major activator of MMP-2. This occurs for both the constitutive expression of MT1-MMP and that up-regulated by angiogenic factors. In either cases, reduction of MT1-MMP levels by IDV is preceded by the inhibition of the binding of the specificity protein (Sp)1 transcription factor to the promoter region of the MT1-MMP gene in endothelial cell nuclei. As MT1-MMP is key for tumour angiogenesis, these results support the use of IDV or its derivatives in anti-cancer therapy. This is recommended by the low toxicity of the drug, and the large body of data on its pharmacokinetic.
Assuntos
Células Endoteliais/metabolismo , Regulação Enzimológica da Expressão Gênica , Inibidores da Protease de HIV/química , Indinavir/farmacologia , Metaloproteinase 14 da Matriz/metabolismo , Animais , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Nus , Neovascularização Patológica , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição Sp1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
MicroRNA (miRs) represent a class of small non-coding regulatory RNAs playing a major role in the control of gene expression by repressing protein synthesis at the post-transcriptional level. Studies carried out during the last years have shown that some miRNAs plays a key role in the control of normal and malignant hgematopoiesis. In this review we focus on recent progress in analyzing the functional role of miR-146a in the control of normal and malignant hematopoiesis. On the other hand, this miRNA has shown to impact in the control of innate immune responses. Finally, many recent studies indicate a deregulation of miR-146 in many solid tumors and gene knockout studies indicate a role for this miRNA as a tumor suppressor.
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Hematopoese/genética , Imunidade/genética , MicroRNAs/fisiologia , Neoplasias/genética , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Genes Supressores de Tumor/fisiologia , Humanos , Sistema Imunitário/metabolismo , Sistema Imunitário/fisiologia , Imunidade/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/imunologiaRESUMO
Although the triterpene CDDO and its potent derivatives, CDDO-Im and CDDO-Me, are now in phase I/II studies in the treatment of some pathological conditions, their effects on normal hematopoiesis are not known. In the present study we provide evidence that CDDO-Im exerts in vitro a potent inhibitory effect on erythroid cell proliferation and survival and a stimulatory action on megakaryocytic differentiation. The effect of CDDO-Im on erythroid and megakaryocytic differentiation was evaluated both on normal hemopoietic progenitor cells (HPCs) induced to selective erythroid (E) or megakaryocytic (Mk) differentiation and on erythroleukemic cell lines HEL and TF1. The inhibitory effect of CDDO-Im on erythroid cell survival and proliferation is mainly related to a reduced GATA-1 expression. This conclusion is supported by the observation that GATA-1 overexpressing TF1 cells are partially protected from the inhibitory effect of CDDO-Im on cell proliferation and survival. The stimulatory effect of CDDO-Im on normal megakaryopoiesis is seemingly related to upmodulation of GATA2 expression and induction of mitogen-activated protein kinases ERK1/2.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Imidazóis/farmacologia , Megacariócitos/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Antígenos CD34/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Citometria de Fluxo , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Integrina beta3/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Megacariócitos/citologia , Megacariócitos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Ácido Oleanólico/farmacologia , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Fatores de TempoRESUMO
alpha and beta dystrobrevins are cytoplasmic components of the dystrophin-associated protein complex that are thought to play a role as scaffold proteins in signal transduction and intracellular transport. In the search of new insights into the functions of beta-dystrobrevin, the isoform restricted to non-muscle tissues, we performed a two-hybrid screen of a mouse cDNA library to look for interacting proteins. Among the positive clones, one encodes iBRAF/HMG20a, a high mobility group (HMG)-domain protein that activates REST (RE-1 silencing transcription factor)-responsive genes, playing a key role in the initiation of neuronal differentiation. We characterized the beta-dystrobrevin-iBRAF interaction by in vitro and in vivo association assays, localized the binding region of one protein to the other, and assessed the kinetics of the interaction as one of high affinity. We also found that beta-dystrobrevin directly binds to BRAF35/HMG20b, a close homologue of iBRAF and a member of a co-repressor complex required for the repression of neural specific genes in neuronal progenitors. In vitro assays indicated that beta-dystrobrevin binds to RE-1 and represses the promoter activity of synapsin I, a REST-responsive gene that is a marker for neuronal differentiation. Altogether, our data demonstrate a direct interaction of beta-dystrobrevin with the HMG20 proteins iBRAF and BRAF35 and suggest that beta-dystrobrevin may be involved in regulating chromatin dynamics, possibly playing a role in neuronal differentiation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Associadas à Distrofina/fisiologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Neurônios/citologia , Animais , Células COS , Proteínas de Ciclo Celular , Diferenciação Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Cromatina/química , Humanos , Cinética , Camundongos , Distrofias Musculares/metabolismo , Ratos , Ressonância de Plasmônio de SuperfícieRESUMO
MicroRNAs (miRNAs) are a class of non-coding protein, single-stranded RNA of 18-22 nucleotides, that exert their actions at post-transcriptional level, mostly through base pairing with the 3'-untranslated region of the target mRNA, thus leading to its translational repression and/or degradation. Recent studies have shown that miRNAs play a crucial role in normal hematopoiesis through the control of the expression of key regulators of hematopoiesis (i.e., transcription factors, growth factor receptors, chemokine receptors), involving regulatory loops that selectively operate in the various hematopoietic lineages. Extensive miRNA deregulation has been observed in leukemia and functional studies support a role for miRNAs in the pathogenesis of these disorders.
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MicroRNAs/genética , Mielopoese/genética , Regiões 3' não Traduzidas , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Processamento Pós-Transcricional do RNARESUMO
MicroRNAs (miRNAs) are noncoding small RNAs that repress protein translation by targeting specific messenger RNAs. miR-15a and miR-16-1 act as putative tumor suppressors by targeting the oncogene BCL2. These miRNAs form a cluster at the chromosomal region 13q14, which is frequently deleted in cancer. Here, we report that the miR-15a and miR-16-1 cluster targets CCND1 (encoding cyclin D1) and WNT3A, which promotes several tumorigenic features such as survival, proliferation and invasion. In cancer cells of advanced prostate tumors, the miR-15a and miR-16 level is significantly decreased, whereas the expression of BCL2, CCND1 and WNT3A is inversely upregulated. Delivery of antagomirs specific for miR-15a and miR-16 to normal mouse prostate results in marked hyperplasia, and knockdown of miR-15a and miR-16 promotes survival, proliferation and invasiveness of untransformed prostate cells, which become tumorigenic in immunodeficient NOD-SCID mice. Conversely, reconstitution of miR-15a and miR-16-1 expression results in growth arrest, apoptosis and marked regression of prostate tumor xenografts. Altogether, we propose that miR-15a and miR-16 act as tumor suppressor genes in prostate cancer through the control of cell survival, proliferation and invasion. These findings have therapeutic implications and may be exploited for future treatment of prostate cancer.
Assuntos
MicroRNAs/genética , Família Multigênica/genética , Proteínas Oncogênicas/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Humanos , Masculino , Camundongos , Proteínas Oncogênicas/genética , Neoplasias da Próstata/patologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3ARESUMO
MicroRNAs (miRNAs or miRs) regulate diverse normal and abnormal cell functions. We have identified a regulatory pathway in normal megakaryopoiesis, involving the PLZF transcription factor, miR-146a and the SDF-1 receptor CXCR4. In leukaemic cell lines PLZF overexpression downmodulated miR-146a and upregulated CXCR4 protein, whereas PLZF knockdown induced the opposite effects. In vitro assays showed that PLZF interacts with and inhibits the miR-146a promoter, and that miR-146a targets CXCR4 mRNA, impeding its translation. In megakaryopoietic cultures of CD34(+) progenitors, PLZF was upregulated, whereas miR-146a expression decreased and CXCR4 protein increased. MiR-146a overexpression and PLZF or CXCR4 silencing impaired megakaryocytic (Mk) proliferation, differentiation and maturation, as well as Mk colony formation. Mir-146a knockdown induced the opposite effects. Rescue experiments indicated that the effects of PLZF and miR-146a are mediated by miR-146a and CXCR4, respectively. Our data indicate that megakaryopoiesis is controlled by a cascade pathway, in which PLZF suppresses miR-146a transcription and thereby activates CXCR4 translation.
Assuntos
Hematopoese/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Megacariócitos/fisiologia , MicroRNAs/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais/fisiologia , Sequência de Bases , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like/genética , Megacariócitos/citologia , MicroRNAs/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteína com Dedos de Zinco da Leucemia Promielocítica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores CXCR4/genética , Células-Tronco/citologia , Células-Tronco/fisiologia , Transcrição GênicaRESUMO
The promyelocytic leukemia zinc finger (PLZF) protein has been described as a transcriptional repressor of homeobox (HOX)-containing genes during embryogenesis. As we previously demonstrated a functional link between overexpression of HOXB7 and melanoma progression, we investigated the lack of PLZF as the possible cause of HOXB7 constitutive activation in these neoplastic cells. Accordingly, we found PLZF expression in melanocytes, but not in melanoma cells, a pattern inversely related to that of HOXB7. PLZF retroviral gene transduction was then performed in a panel of melanoma cell lines, and tumorigenicity was compared with that of empty vector-transduced control cell lines. Evaluation of in vitro migration, invasion and adhesion indicated that PLZF gene transduction induced a less malignant phenotype, as confirmed through in vivo studies performed in athymic nude mice. This reduced tumorigenicity was not coupled with HOXB7 repression. In order to find more about the molecular targets of PLZF, the gene expression profiles of PLZF- and empty vector-transduced A375 melanoma cells were analysed by Atlas Cancer macroarray. Among several genes modulated by PLZF enforced expression, of particular interest were integrin alphavbeta3, osteonectin/SPARC and matrix metalloprotease-9 that were downmodulated, and the tyrosinase-related protein-1 that was upregulated in all the analysed samples. This profile confirms the reduced tumorigenic phenotype with reversion to a more differentiated, melanocyte like, pattern, thus suggesting a suppressor role for PLZF in solid tumors. Moreover, these results indicate that PLZF and HOXB7 are functionally independent and that their coupled deregulation may account for most of the alterations described in melanomas.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Melanoma/patologia , Glicoproteínas de Membrana , Oxirredutases , Fatores de Transcrição/fisiologia , Animais , Testes de Carcinogenicidade , Divisão Celular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Integrina alfaVbeta3/genética , Fatores de Transcrição Kruppel-Like , Metaloproteinase 9 da Matriz/genética , Melanócitos/metabolismo , Melanoma/genética , Camundongos , Camundongos Nus , Invasividade Neoplásica , Osteonectina/genética , Regiões Promotoras Genéticas , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteínas/genética , Valores de Referência , Células Tumorais CultivadasRESUMO
We investigated the expression of the PLZF gene in purified human hematopoietic progenitors induced to unilineage erythroid, granulocytic or megakaryocytic differentiation and maturation in serum-free culture. PLZF is expressed in quiescent progenitors: the expression level progressively rises through megakaryocytic development, whereas it gradually declines in erythroid and granulopoietic culture. To investigate the role of PLZF in megakaryopoiesis, we transduced the PLZF gene into the erythro-megakaryocytic TF1 cell line. PLZF overexpression upmodulates the megakaryocytic specific markers (CD42a, CD42b, CD61, PF4) and induces the thrombopoietin receptor (TpoR). The proximal promoter of the TpoR gene is activated in PLZF-expressing TF1 cells: in this promoter region, a PLZF DNA-binding site was identified by deletion constructs studies. Interestingly, PLZF and GATA1 proteins coimmunoprecipitate in PLZF-expressing TF1 cells: enforced expression of both PLZF and GATA1 in TF1 cells results in increased upregulation of megakaryocytic markers, as compared to exogenous PLZF or GATA1 alone, suggesting a functional role for the PLZF/GATA1 complex. Our data indicate that PLZF plays a significant stimulatory role in megakaryocytic development, seemingly mediated in part by induction of TpoR expression at transcriptional level. This stimulatory effect is potentiated by physical interaction of PLZF and GATA1, which are possibly assembled in a multiprotein transcriptional complex.