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1.
Haematologica ; 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296574

RESUMO

CD38 is expressed in several types of non-Hodgkin lymphoma and constitutes a promising target for antibody-based therapy. Daratumumab (Darzalex) is a first-in-class anti-CD38 antibody approved for the treatment of relapsed/refractory multiple myeloma. It has also demonstrated clinical activity in Waldenstrom macroglobulinaemia and amyloidosis. Here, we have evaluated the activity and mechanism of action of daratumumab in preclinical in vitro and in vivo models of mantle cell lymphoma, follicular lymphoma and diffuse large B cell lymphoma, as monotherapy or in combination with standard chemo-immunotherapy. In vitro, daratumumab engages Fc-mediated cytotoxicity by antibody-dependent cell cytotoxicity and antibody-dependent cell phagocytosis in all lymphoma subtypes. In the presence of human serum, complement-dependent cell cytotoxicity was marginally engaged. We demonstrated by Selective Plane Illumination Microscopy that daratumumab fully penetrated a 3D lymphoma organoid and decreased organoid volume. In vivo, daratumumab completely prevents tumor outgrowth in models of mantle cell and follicular lymphoma, and shows comparable activity to rituximab in a disseminated in vivo model of blastic mantle cell lymphoma. Moreover, daratumumab improves overall survival in a mouse model of transformed CD20dim follicular lymphoma, where rituximab showed limited activity. Daratumumab potentiates the antitumor activity of CHOP and R-CHOP in mantle cell and follicular lymphoma xenografts. Furthermore, in a patient-derived diffuse large B cell lymphoma xenograft model, daratumumab anti-tumor activity was comparable to R-CHOP and the addition of daratumumab to either CHOP or R-CHOP led to full tumor regression. In summary, daratumumab constitutes a novel therapeutic opportunity in certain scenarios and these results warrant further clinical development.

2.
Haematologica ; 104(5): 881-893, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30923099

RESUMO

Advances in genome engineering in the last decade, particularly in the development of programmable nucleases, have made it possible to edit the genomes of most cell types precisely and efficiently. Chief among these advances, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a novel, versatile and easy-to-use tool to edit genomes irrespective of their complexity, with multiple and broad applications in biomedicine. In this review, we focus on the use of CRISPR/Cas9 genome editing in the context of hematologic diseases and appraise the major achievements and challenges in this rapidly moving field to gain a clearer perspective on the potential of this technology to move from the laboratory to the clinic. Accordingly, we discuss data from studies editing hematopoietic cells to understand and model blood diseases, and to develop novel therapies for hematologic malignancies. We provide an overview of the applications of gene editing in experimental, preclinical and clinical hematology including interrogation of gene function, target identification and drug discovery and chimeric antigen receptor T-cell engineering. We also highlight current limitations of CRISPR/Cas9 and the possible strategies to overcome them. Finally, we consider what advances in CRISPR/Cas9 are needed to move the hematology field forward.

3.
Nucleic Acids Res ; 47(10): 5016-5037, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30923829

RESUMO

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.


Assuntos
Apoptose , Diferenciação Celular , Cromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Células Mieloides/metabolismo , Acetilação , Animais , Células Cultivadas , Cromatina/genética , Epigênese Genética , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/citologia , Processamento de Proteína Pós-Traducional , Transcrição Genética
4.
Haematologica ; 104(4): 778-788, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29954928

RESUMO

Constitutive activation of the chemokine receptor CXCR4 has been associated with tumor progression, invasion, and chemotherapy resistance in different cancer subtypes. Although the CXCR4 pathway has recently been suggested as an adverse prognostic marker in diffuse large B-cell lymphoma, its biological relevance in this disease remains underexplored. In a homogeneous set of 52 biopsies from patients, an antibody-based cytokine array showed that tissue levels of CXCL12 correlated with high microvessel density and bone marrow involvement at diagnosis, supporting a role for the CXCL12-CXCR4 axis in disease progression. We then identified the tetra-amine IQS-01.01RS as a potent inverse agonist of the receptor, preventing CXCL12-mediated chemotaxis and triggering apoptosis in a panel of 18 cell lines and primary cultures, with superior mobilizing properties in vivo than those of the standard agent. IQS-01.01RS activity was associated with downregulation of p-AKT, p-ERK1/2 and destabilization of MYC, allowing a synergistic interaction with the bromodomain and extra-terminal domain inhibitor, CPI203. In a xenotransplant model of diffuse large B-cell lymphoma, the combination of IQS-01.01RS and CPI203 decreased tumor burden through MYC and p-AKT downregulation, and enhanced the induction of apoptosis. Thus, our results point out an emerging role of CXCL12-CXCR4 in the pathogenesis of diffuse large B-cell lymphoma and support the simultaneous targeting of CXCR4 and bromodomain proteins as a promising, rationale-based strategy for the treatment of this disease.

5.
Nature ; 554(7690): 106-111, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29298288

RESUMO

Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells, securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations. HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality. Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs, a finding that is supported by studies of unperturbed haematopoiesis and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs. Other studies suggested that such lineage bias might reflect generation of unipotent or oligopotent self-renewing progenitors within the phenotypic HSC compartment, and implicated uncoupling of the defining HSC properties of self-renewal and multipotency. Here we use highly sensitive tracking of progenitors and mature cells of the megakaryocyte/platelet, erythroid, myeloid and B and T cell lineages, produced from singly transplanted HSCs, to reveal a highly organized, predictable and stable framework for lineage-restricted fates of long-term self-renewing HSCs. Most notably, a distinct class of HSCs adopts a fate towards effective and stable replenishment of a megakaryocyte/platelet-lineage tree but not of other blood cell lineages, despite sustained multipotency. No HSCs contribute exclusively to any other single blood-cell lineage. Single multipotent HSCs can also fully restrict towards simultaneous replenishment of megakaryocyte, erythroid and myeloid lineages without executing their sustained lymphoid lineage potential. Genetic lineage-tracing analysis also provides evidence for an important role of platelet-biased HSCs in unperturbed adult haematopoiesis. These findings uncover a limited repertoire of distinct HSC subsets, defined by a predictable and hierarchical propensity to adopt a fate towards replenishment of a restricted set of blood lineages, before loss of self-renewal and multipotency.


Assuntos
Linhagem da Célula , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Multipotentes/citologia , Animais , Antígenos CD34 , Linfócitos B/citologia , Plaquetas/citologia , Antígeno CD48/deficiência , Autorrenovação Celular , Células Eritroides/citologia , Feminino , Células-Tronco Hematopoéticas/metabolismo , Masculino , Megacariócitos/citologia , Camundongos , Células-Tronco Multipotentes/metabolismo , Células Mieloides/citologia , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Linfócitos T/citologia
7.
Oncotarget ; 8(39): 66742-66746, 2017 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-29112206

RESUMO

This meeting held at the University of Barcelona in March 2017, brought together scientists and clinicians worldwide to discuss current and future clinico-biological implications of intratumoral heterogeneity (ITH) and subclonal evolution in cancer diagnosis, patient stratification, and treatment resistance in diagnosis, treatment and follow-up. There was consensus that both longitudinal and tumor multi-region studies in matched samples are needed to better understand the dynamics of ITH. The contribution of the epigenome and microenvironment to ITH and subclone evolution remains understudied. It was recommended to combine computational, pathology and imaging tools to study the role of the microenvironment in subclone selection/evolution.

8.
Sci Rep ; 7(1): 13946, 2017 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-29066743

RESUMO

Mantle cell lymphoma (MCL) is a hematologic neoplasm characterised by the t(11;14)(q13;q32) translocation leading to aberrant cyclin D1 expression. The cell functions of cyclin D1 depend on its partners and/or subcellular distribution, resulting in different oncogenic properties. We observed the accumulation of cyclin D1 in the cytoplasm of a subset of MCL cell lines and primary cells. In primary cells, this cytoplasmic distribution was correlated with a more frequent blastoid phenotype. We performed immunoprecipitation assays and mass spectrometry on enriched cytosolic fractions from two cell lines. The cyclin D1 interactome was found to include several factors involved in adhesion, migration and invasion. We found that the accumulation of cyclin D1 in the cytoplasm was associated with higher levels of migration and invasiveness. We also showed that MCL cells with high cytoplasmic levels of cyclin D1 engrafted more rapidly into the bone marrow, spleen, and brain in immunodeficient mice. Both migration and invasion processes, both in vivo and in vitro, were counteracted by the exportin 1 inhibitor KPT-330, which retains cyclin D1 in the nucleus. Our data reveal a role of cytoplasmic cyclin D1 in the control of MCL cell migration and invasion, and as a true operator of MCL pathogenesis.


Assuntos
Movimento Celular , Ciclina D1/metabolismo , Citoplasma/metabolismo , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/patologia , Transporte Ativo do Núcleo Celular , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Núcleo Celular/metabolismo , Transformação Celular Neoplásica , Citosol/metabolismo , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Proteômica
9.
Nat Commun ; 7: 11075, 2016 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-27009448

RESUMO

Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis.


Assuntos
Plaquetas/metabolismo , Senescência Celular , Células-Tronco Hematopoéticas/citologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Linhagem da Célula/genética , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Células Mieloides/citologia , Proteínas Nucleares/metabolismo , Fenótipo , Fatores de Transcrição/metabolismo
10.
Stem Cells Dev ; 25(3): 259-65, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26603126

RESUMO

Hematopoietic stem cell (HSC) engraftment has been achieved using single-cell transplantation of prospectively highly purified adult HSC populations. However, bulk transplants are still performed when assessing the HSC potential of early embryonic hematopoietic tissues such as the aorta-gonad mesonephros (AGM) due to very low HSC activity content early in development. Intra-bone marrow transplantation (IBMT) has emerged as a superior administration route over intravenous (IV) transplantation for assessing the reconstituting ability of human HSCs in the xenotransplant setting since it bypasses the requirement for homing to the BM. In this study, we compared the ability of IBMT and IV administration of embryonic day 11.5 AGM-derived cells to reconstitute the hematopoietic system of myeloablated recipients. IBMT resulted in higher levels of AGM HSC long-term multilineage engraftment in the peripheral blood, BM, spleen, and thymus of primary and secondary recipients, and in limiting dilution experiments. The administration route did not skew the multilineage contribution pattern, but IBMT conferred higher Lineage(-)Sca-1(+)c-kit(+) long-term engraftment, in line with the superior IBMT reconstitution. Therefore, IBMT represents a superior administration route to detect HSC activity from developmentally early sources with limited HSC activity content, such as the AGM.


Assuntos
Transplante de Medula Óssea/métodos , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Transplante de Células-Tronco Hematopoéticas/métodos , Mesonefro/citologia , Animais , Aorta/citologia , Aorta/embriologia , Células da Medula Óssea/citologia , Células Cultivadas , Células-Tronco Embrionárias/transplante , Gônadas/citologia , Gônadas/embriologia , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Timo/citologia
11.
Blood ; 126(25): 2676-85, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26463423

RESUMO

Infant B-cell acute lymphoblastic leukemia (B-ALL) accounts for 10% of childhood ALL. The genetic hallmark of most infant B-ALL is chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene. Despite improvement in the clinical management and survival (∼85-90%) of childhood B-ALL, the outcome of infants with MLL-rearranged (MLL-r) B-ALL remains dismal, with overall survival <35%. Among MLL-r infant B-ALL, t(4;11)+ patients harboring the fusion MLL-AF4 (MA4) display a particularly poor prognosis and a pro-B/mixed phenotype. Studies in monozygotic twins and archived blood spots have provided compelling evidence of a single cell of prenatal origin as the target for MA4 fusion, explaining the brief leukemia latency. Despite its aggressiveness and short latency, current progress on its etiology, pathogenesis, and cellular origin is limited as evidenced by the lack of mouse/human models recapitulating the disease phenotype/latency. We propose this is because infant cancer is from an etiologic and pathogenesis standpoint distinct from adult cancer and should be seen as a developmental disease. This is supported by whole-genome sequencing studies suggesting that opposite to the view of cancer as a "multiple-and-sequential-hit" model, t(4;11) alone might be sufficient to spawn leukemia. The stable genome of these patients suggests that, in infant developmental cancer, one "big-hit" might be sufficient for overt disease and supports a key contribution of epigenetics and a prenatal cell of origin during a critical developmental window of stem cell vulnerability in the leukemia pathogenesis. Here, we revisit the biology of t(4;11)+ infant B-ALL with an emphasis on its origin, genetics, and disease models.


Assuntos
Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Translocação Genética/genética , Animais , Humanos , Lactente , Fatores de Elongação da Transcrição
12.
Nature ; 502(7470): 232-6, 2013 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-23934107

RESUMO

The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding--a common and life-threatening side effect of many cancer therapies--and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output.


Assuntos
Plaquetas/citologia , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Animais , Linhagem da Célula/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Linfócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
13.
Nat Immunol ; 13(4): 412-9, 2012 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-22344248

RESUMO

The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage-commitment process transits from the bone marrow to the remote thymus.


Assuntos
Linfócitos B/citologia , Linhagem da Célula/imunologia , Células Progenitoras Linfoides/citologia , Células Mieloides/citologia , Células Precursoras de Linfócitos B/citologia , Linfócitos T/citologia , Animais , Separação Celular , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Células Progenitoras Linfoides/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Timo/citologia
14.
EMBO J ; 31(2): 351-65, 2012 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-22068055

RESUMO

The transcription factors that control lineage specification of haematopoietic stem cells (HSCs) have been well described for the myeloid and lymphoid lineages, whereas transcriptional control of erythroid (E) and megakaryocytic (Mk) fate is less understood. We here use conditional removal of the GATA-1 and FOG-1 transcription factors to identify FOG-1 as required for the formation of all committed Mk- and E-lineage progenitors, whereas GATA-1 was observed to be specifically required for E-lineage commitment. FOG-1-deficient HSCs and preMegEs, the latter normally bipotent for the Mk and E lineages, underwent myeloid transcriptional reprogramming, and formed myeloid, but not erythroid and megakaryocytic cells in vitro. These results identify FOG-1 and GATA-1 as required for formation of bipotent Mk/E progenitors and their E-lineage commitment, respectively, and show that FOG-1 mediates transcriptional Mk/E programming of HSCs as well as their subsequent Mk/E-lineage commitment. Finally, C/EBPs and FOG-1 exhibited transcriptional cross-regulation in early myelo-erythroid progenitors making their functional antagonism a potential mechanism for separation of the myeloid and Mk/E lineages.


Assuntos
Eritropoese/genética , Fator de Transcrição GATA1/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Proteínas Nucleares/fisiologia , Trombopoese/genética , Fatores de Transcrição/fisiologia , Animais , Células da Medula Óssea/citologia , Proteína beta Intensificadora de Ligação a CCAAT/deficiência , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/deficiência , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem da Célula , Células Cultivadas/citologia , Ensaio de Unidades Formadoras de Colônias , Células Precursoras Eritroides/citologia , Fator de Transcrição GATA1/genética , Células Progenitoras de Megacariócitos/citologia , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Transcrição Genética
15.
Free Radic Biol Med ; 42(12): 1766-80, 2007 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-17512456

RESUMO

Lipoic acid (LA) is a widely used antioxidant that protects mitochondria from oxidative damage in vivo. Much of this protection is thought to be due to the reduction of LA to dihydrolipoic acid (LAH(2)). This reduction is catalyzed in vivo by thioredoxin, thioredoxin reductase (TrxR), and lipoamide dehydrogenase. We hypothesized that specifically targeting LA to mitochondria, the site of most cellular reactive oxygen species production, would make it a more effective antioxidant. To do this, we made a novel molecule, MitoLipoic acid, by attaching lipoic acid to the lipophilic triphenylphosphonium cation. MitoL was accumulated rapidly within mitochondria several-hundred fold driven by the membrane potential. MitoL was reduced to the active antioxidant dihydroMitoLipoic acid by thioredoxin and by lipoamide dehydrogenase but not by TrxR. In isolated mitochondria or cells MitoL was only slightly reduced (5-10%), while, in contrast, LA was extensively reduced. This difference was largely due to the reaction of LA with TrxR, which did not occur for MitoL. Furthermore, in cells MitoL was quantitatively converted to an S-methylated product. As a consequence of its lack of reduction, MitoL was not protective for mitochondria or cells against a range of oxidative stresses. These results suggest that the protective action of LA in vivo may require its reduction to LAH(2) and that this reduction is largely mediated by TrxR.


Assuntos
Antioxidantes/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Compostos Organofosforados/síntese química , Ácido Tióctico/farmacologia , Animais , Antioxidantes/química , Di-Hidrolipoamida Desidrogenase/metabolismo , Mitocôndrias Hepáticas/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Oxirredução , Estresse Oxidativo , Ratos , Espécies Reativas de Oxigênio , Ácido Tióctico/química , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo
16.
Cancer Lett ; 233(1): 131-8, 2006 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-16473674

RESUMO

Cellular response to limiting oxygen levels is managed, in part, by the transcription factor hypoxia-inducible factor 1 (HIF-1), and the prolyl hydroxylase (PHD) family of oxygen-requiring enzymes. In the process of analyzing the expression of PHD3, we observed the presence of two alternatively processed PHD3 transcripts, designated PHD3Delta1 and PHD3Delta4 . The expression of both PHD3 and PHD3Delta1 was observed in all tissues and cell lines tested, although the expression of the novel PHD3Delta4 appeared to be restricted to primary cancer tissues. The function of PHD3Delta4 was assessed in transfection experiments showing a preserved prolyl hydroxylase activity. We would submit that PHD3 variants generated by alternative splicing may be intrinsically involved in the complex system of oxygen sensing.


Assuntos
Pró-Colágeno-Prolina Dioxigenase/genética , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Dioxigenases , Humanos , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Dados de Sequência Molecular , Pró-Colágeno-Prolina Dioxigenase/química , Pró-Colágeno-Prolina Dioxigenase/metabolismo
17.
FEBS Lett ; 579(12): 2669-74, 2005 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-15862307

RESUMO

Exposure to limiting oxygen in cells and tissues induce the stabilization and transcriptional activation of the hypoxia-inducible factor 1 alpha (HIF-1alpha) protein, a key regulator of the hypoxic response. Reactive oxygen species (ROS) generation has been implicated in the stabilization of HIF-1alpha during this response, but this is still a matter of some debate. In this study we utilize a mitochondria-targeted antioxidant, mitoubiquinone (MitoQ), and examine its effects on the hypoxic stabilization of HIF-1alpha. Our results show that under conditions of reduced oxygen (3% O(2)), MitoQ ablated the hypoxic induction of ROS generation and destabilized HIF-1alpha protein. This in turn led to an abrogation of HIF-1 transcriptional activity. Normoxic stabilization of HIF-1alpha, on the other hand, was unchanged in the presence of MitoQ suggesting that ROS were not involved. This study strongly suggests that mitochondrial ROS contribute to the hypoxic stabilization of HIF-1alpha.


Assuntos
Antioxidantes/metabolismo , Hipóxia Celular , Mitocôndrias/química , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/metabolismo , Western Blotting , Linhagem Celular Tumoral , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Expressão Gênica , Marcação de Genes , Genes Reporter , Hepatoblastoma/metabolismo , Hepatoblastoma/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Luciferases/metabolismo , Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
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