RESUMO
Haematopoiesis is a paradigm of cell differentiation because of the wide variety and overwhelming number of mature blood cells produced daily. Under stress conditions, the organism must adapt to a boosted demand for blood cells. Chronic granulomatous disease (CGD) is a genetic disease caused by inactivating mutations that affect the phagocyte oxidase. Besides a defective innate immune system, CGD patients suffer from recurrent hyper-inflammation episodes, circumstances upon which they must face emergency haematopoiesis. The targeting of Cybb and Ncf1 genes have produced CGD animal models that are a useful surrogate when studying the pathophysiology and treatment of this disease. Here, we show that Cyba-/- mice spontaneously develop granuloma and, therefore, constitute a CGD animal model to complement the existing Cybb-/- and Ncf1-/- models. More importantly, we have analysed haematopoiesis in granuloma-bearing Cyba-/- mice. These animals showed a significant loss of weight, developed remarkable splenomegaly, bone marrow myeloid hyperplasia, and signs of anaemia. Haematological analyses showed a sharped decrease of B-cells and a striking development of myeloid cells in all compartments. Collectively, our results show that granuloma inflammatory lesions dramatically change haematopoiesis homeostasis. Consequently, we suggest that besides their defective innate immunity, the alteration of haematopoiesis homeostasis upon granuloma may contribute to the dismal outcome of CGD.
Assuntos
Linfócitos B/metabolismo , Grupo dos Citocromos b/genética , Doença Granulomatosa Crônica/patologia , Células Mieloides/patologia , NADPH Oxidases/genética , Animais , Sistemas CRISPR-Cas , Linhagem da Célula , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/imunologia , Humanos , Hiperplasia , Masculino , Camundongos , Células Mieloides/imunologiaRESUMO
The regulation of protein function by reversible oxidation is increasingly recognized as a key mechanism for the control of cellular signaling, modulating crucial biological processes such as cell differentiation. In this scenario, NADPH oxidases must occupy a prominent position. Our results show that hematopoietic stem and progenitor cells express three p22phox-dependent NADPH oxidases members (NOX1, NOX2 and NOX4). By deleting the p22phox coding gene (Cyba), here we have analyzed the importance of this family of enzymes during in vivo hematopoiesis. Cyba-/- mice show a myeloid bias, and an enrichment of hematopoietic stem cell populations. By means of hematopoietic transplant experiments we have also tried to dissect the specific role of the NADPH oxidases. While the absence of NOX1 or NOX2 provides a higher level of reconstitution, a lack of NOX4 rendered the opposite result, suggesting a functional specificity among the different NADPH oxidases. Cyba-/- cells showed a hampered activation of AKT1 and a sharp decrease in STAT5 protein. This is in line with the diminished response to IL-7 shown by our results, which could explain the overproduction of immunoglobulins observed in Cyba-/- mice.
Assuntos
Imunoglobulinas , NADPH Oxidases , Animais , Células-Tronco Hematopoéticas , Camundongos , Camundongos Knockout , NADPH Oxidase 4 , NADPH Oxidases/genética , Espécies Reativas de OxigênioRESUMO
BACKGROUND: The differentiation-based therapy for acute promyelocytic leukemia (APL) is an inspiring example for the search of novel strategies aimed at treatment of other subtypes of acute myeloid leukemia (AML). Thus, the discovery of new molecular players in cell differentiation becomes a paramount research area to achieve this goal. Here, the involvement of the protein tyrosine phosphatases SHP1 and SHP2 on leukemic cells differentiation is shown, along with the therapeutic possibilities of their targeting to enhance the differentiation induction effect of phorbol esters. METHODS: The oxidation status and enzymatic activity of SHP1 and SHP2 during PMA-induced differentiation of HEL cells was evaluated. Additionally, the effects of RNAi-mediated downregulation of these phosphatases on cell differentiation was studied. Afterwards, the impact of chemical inhibition of SHP1 and SHP2 on differentiation both in the presence and absence of phorbol esters was tested. Finally, the anti-leukemic potential of phorbol esters and chemical inhibitors of SHP1 and SHP2 was addressed in several AML model cell lines, a xenograft mouse model and AML primary cells in vitro. RESULTS: An increase of oxidation with a concomitant decrease of activity was observed for both phosphatases at the onset of PMA-induced differentiation. Consistently, silencing of these proteins favored the process, with an enhanced effect upon their simultaneous downregulation. Moreover, the proteins SRC and ß-catenin were identified as downstream targets of SHP1 and SHP2 in this context. In agreement with these findings, chemical inhibition of the phosphatases promoted cell differentiation itself and enhanced the effect of phorbol esters. Interestingly, treatment with the phorbol ester prostratin and the dual inhibitor of SHP1 and SHP2 NSC87877 synergistically hampered the proliferation of AML cell lines, prolonged the survival of xenografted mice and reduced the clonogenic potential of AML primary cells. CONCLUSIONS: SHP1 and SHP2 are relevant mediators of differentiation in AML cells and their inhibition either alone or in combination with prostratin seems a promising differentiation-based therapeutic strategy against different subtypes of AML beyond APL.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , Ésteres de Forbol/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Animais , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oxidative stress is related to ageing and degenerative diseases, including cancer. However, a moderate amount of reactive oxygen species (ROS) is required for the regulation of cellular signalling and gene expression. A low level of ROS is important for maintaining quiescence and the differentiation potential of haematopoietic stem cells (HSCs), whereas the level of ROS increases during haematopoietic differentiation; thus, suggesting the importance of redox signalling in haematopoiesis. Here, we will analyse the importance of ROS for haematopoiesis and include evidence showing that cells from leukaemia patients live under oxidative stress. The potential sources of ROS will be described. Finally, the level of oxidative stress in leukaemic cells can also be harnessed for therapeutic purposes. In this regard, the reliance of front-line anti-leukaemia chemotherapeutics on increased levels of ROS for their mechanism of action, as well as the active search for novel compounds that modulate the redox state of leukaemic cells, will be analysed.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Humanos , Leucemia/tratamento farmacológico , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The regulation of hematopoietic stem cells (HSCs) depends on the integration of the multiple signals received from the bone marrow niche. We show the relevance of the protein tyrosine phosphatase PTPN13 and ß-catenin as intracellular signaling molecules to control HSCs adhesiveness, cell cycling, and quiescence. Lethally irradiated mice transplanted with Lin(-) bone marrow cells in which PTPN13 or ß-catenin had been silenced showed a significant increase of long-term (LT) and short-term (ST) HSCs. A decrease in cycling cells was also found, together with an increase in quiescence. The decreased expression of PTPN13 or ß-catenin was linked to the upregulation of several genes coding for integrins and several cadherins, explaining the higher cell adhesiveness. Our data are consistent with the notion that the levels of PTPN13 and ß-catenin must be strictly regulated by extracellular signaling to regulate HSC attachment to the niche and the balance between proliferation and quiescence.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Hematopoéticas/citologia , Linfopoese , Proteína Tirosina Fosfatase não Receptora Tipo 13/metabolismo , Trombopoese , beta Catenina/metabolismo , Animais , Células da Medula Óssea/metabolismo , Adesão Celular , Comunicação Celular , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Tirosina Fosfatase não Receptora Tipo 13/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Nicho de Células-Tronco , beta Catenina/genéticaRESUMO
PURPOSE: Cancer cells show higher levels of reactive oxygen species (ROS) than normal cells and increasing intracellular ROS levels are becoming a recognized strategy against tumor cells. Thus, diminishing ROS levels could be also detrimental to cancer cells. We surmise that avoiding ROS generation would be a better option than quenching ROS with antioxidants. Chronic myelogenous leukemia (CML) is triggered by the expression of BCR-ABL kinase, whose activity leads to increased ROS production, partly through NADPH oxidases. Here, we assessed NADPH oxidases as therapeutic targets in CML. EXPERIMENTAL DESIGN: We have analyzed the effect of different NADPH oxidase inhibitors, either alone or in combination with BCR-ABL inhibitors, in CML cells and in two different animal models for CML. RESULTS: NADPH oxidase inhibition dramatically impaired the proliferation and viability of BCR-ABL-expressing cells due to the attenuation of BCR-ABL signaling and a pronounced cell-cycle arrest. Moreover, the combination of NADPH oxidase inhibitors with BCR-ABL inhibitors was highly synergistic. Two different animal models underscore the effectiveness of NADPH oxidase inhibitors and their combination with BCR-ABL inhibitors for CML targeting in vivo. CONCLUSION: Our results offer further therapeutic opportunities for CML, by targeting NADPH oxidases. In the future, it would be worthwhile conducting further experiments to ascertain the feasibility of translating such therapies to clinical practice.