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1.
Methods Mol Biol ; 1510: 375-385, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27761836

RESUMEN

The differentiation of hematopoietic stem cells into mature blood cells is a highly ordered process and dysregulation of this process can lead to leukemogenesis. Agents that are used to cure acute promyelocytic leukemia (APL) can induce differentiation and/or apoptosis. Here, we describe how effects of all-trans retinoic acid (ATRA) and histone deacetylase inhibitors (HDACi) on APL cell differentiation can be evaluated by immunoblotting and by flow cytometry. We show how the levels of differentiation-associated transcription factors of the CCAAT enhancer binding protein (C/EBP) family can be determined by Western blot and we explain how the cell surface expression of the leukocyte surface antigen CD11b can be measured by flow cytometry.


Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteínas Potenciadoras de Unión a CCAAT/antagonistas & inhibidores , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Diferenciación Celular/efectos de los fármacos , Citometría de Flujo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Ácidos Hidroxámicos/farmacología , Immunoblotting , Indoles/farmacología , Panobinostat , Piridinas/farmacología , Tretinoina/farmacología
2.
Arch Toxicol ; 91(5): 2191-2208, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-27807597

RESUMEN

The treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) induces granulocytic differentiation. This process renders APL cells resistant to cytotoxic chemotherapies. Epigenetic regulators of the histone deacetylases (HDACs) family, which comprise four classes (I-IV), critically control the development and progression of APL. We set out to clarify the parameters that determine the interaction between ATRA and histone deacetylase inhibitors (HDACi). Our assays included drugs against class I HDACs (MS-275, VPA, and FK228), pan-HDACi (LBH589, SAHA), and the novel HDAC6-selective compound Marbostat-100. We demonstrate that ATRA protects APL cells from cytotoxic effects of SAHA, MS-275, and Marbostat-100. However, LBH589 and FK228, which have a superior substrate-inhibitor dissociation constant (Ki) for the class I deacetylases HDAC1, 2, 3, are resistant against ATRA-dependent cytoprotective effects. We further show that HDACi evoke DNA damage, measured as induction of phosphorylated histone H2AX and by the comet assay. The ability of ATRA to protect APL cells from the induction of p-H2AX by HDACi is a readout for the cytoprotective effects of ATRA. Moreover, ATRA increases the fraction of cells in the G1 phase, together with an accumulation of the cyclin-dependent kinase inhibitor p21 and a reduced expression of thymidylate synthase (TdS). In contrast, the ATRA-dependent activation of the transcription factors STAT1, NF-κB, and C/EBP hardly influences the responses of APL cells to HDACi. We conclude that the affinity of HDACi for class I HDACs determines whether such drugs can kill naïve and maturated APL cells.


Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Leucemia/tratamiento farmacológico , Leucemia/patología , Tretinoina/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Leucemia/metabolismo , FN-kappa B/metabolismo , Piridinas/farmacología , Factor de Transcripción STAT1/metabolismo , Tretinoina/administración & dosificación
3.
Cell Commun Signal ; 13: 40, 2015 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-26369790

RESUMEN

BACKGROUND: Pasteurella multocida toxin (PMT) is a potent inducer of osteoclast formation. Pigs suffering from an infection with toxigenic Pasteurella multocida strains develop atrophic rhinitis characterised by a loss of turbinate bones and conchae. However, on the molecular level the process of bone loss remains largely uncharacterised. RESULTS: Recently it was found that PMT activates the serine/threonine kinase mammalian target of rapamycin (mTOR) in fibroblasts. Using RAW264.7 macrophages, we investigated the role of the mTOR complex 1 (mTORC1) in PMT-mediated osteoclast formation. PMT induces the differentiation of RAW264.7 macrophages into multinucleated, tartrate resistant acid phosphatase (TRAP) positive osteoclasts that are capable to resorb bone. In the presence of the mTORC1 inhibitor rapamycin, PMT was significantly less able to induce the formation of TRAP-positive osteoclasts. Accordingly, the resulting resorption of bone was strongly reduced. A major target of mTOR is the 70 kDa ribosomal protein S6 kinase 1 (p70 S6K1). Activated p70 S6K1 decreases the expression of programmed cell death protein 4 (PDCD4), a negative transcriptional regulator of osteoclastogenesis, at the protein and gene level. Ultimately this results in the activation of c-Jun, a component of the activator protein 1 (AP-1) complex, which is a major transcription factor for the induction of osteoclast-specific genes. We now demonstrate that c-Jun and its downstream target, the osteoclast-specific bone degrading protease cathepsin K, are upregulated upon PMT treatment in an mTOR-dependent manner. CONCLUSIONS: Activation of mTOR signalling plays a central role in the formation of osteoclasts through the bacterial toxin PMT. On the molecular level, PMT-induced activation of mTOR leads to down regulation of PDCD4, a known repressor of AP-1 complex, culminating in the activation of c-Jun, an essential transcription factor for triggering osteoclastogenesis.


Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Resorción Ósea/veterinaria , Macrófagos/microbiología , Complejos Multiproteicos/metabolismo , Osteoclastos/microbiología , Infecciones por Pasteurella/veterinaria , Pasteurella multocida/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Resorción Ósea/metabolismo , Resorción Ósea/microbiología , Resorción Ósea/patología , Catepsina K/metabolismo , Línea Celular , Macrófagos/metabolismo , Macrófagos/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Osteoclastos/metabolismo , Osteoclastos/patología , Infecciones por Pasteurella/complicaciones , Infecciones por Pasteurella/metabolismo , Infecciones por Pasteurella/patología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Porcinos , Factor de Transcripción AP-1/metabolismo
4.
Oncotarget ; 5(8): 2305-17, 2014 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-24810717

RESUMEN

Signal Transducer and Activator of Transcription-1 (STAT1) is phosphorylated upon interferon (IFN) stimulation, which can restrict cell proliferation and survival. Nevertheless, in some cancers STAT1 can act in an anti-apoptotic manner. Moreover, certain malignancies are characterized by the overexpression and constitutive activation of STAT1. Here, we demonstrate that the treatment of transformed hematopoietic cells with epigenetic drugs belonging to the class of histone deacetylase inhibitors (HDACi) leads to the cleavage of STAT1 at multiple sites by caspase-3 and caspase-6. This process does not occur in solid tumor cells, normal hematopoietic cells, and leukemic cells that underwent granulocytic or monocytic differentiation. STAT1 cleavage was studied under cell free conditions with purified STAT1 and a set of candidate caspases as well as with mass spectrometry. These assays indicate that unmodified STAT1 is cleaved at multiple sites by caspase-3 and caspase-6. Our study shows that STAT1 is targeted by caspases in malignant undifferentiated hematopoietic cells. This observation may provide an explanation for the selective toxicity of HDACi against rapidly proliferating leukemic cells.


Asunto(s)
Caspasa 3/metabolismo , Caspasa 6/metabolismo , Leucemia/metabolismo , Factor de Transcripción STAT1/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Western Blotting , Butiratos/farmacología , Línea Celular Tumoral , Citometría de Flujo , Inhibidores de Histona Desacetilasas/farmacología , Humanos
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