RESUMEN
Enforced expression of microRNA-155 (miR-155) in myeloid cells has been shown to have both oncogenic or tumour-suppressor functions in acute myeloid leukaemia (AML). We sought to resolve these contrasting effects of miR-155 overexpression using murine models of AML and human paediatric AML data sets. We show that the highest miR-155 expression levels inhibited proliferation in murine AML models. Over time, enforced miR-155 expression in AML in vitro and in vivo, however, favours selection of intermediate miR-155 expression levels that results in increased tumour burden in mice, without accelerating the onset of disease. Strikingly, we show that intermediate and high miR-155 expression also regulate very different subsets of miR-155 targets and have contrasting downstream effects on the transcriptional environments of AML cells, including genes involved in haematopoiesis and leukaemia. Furthermore, we show that elevated miR-155 expression detected in paediatric AML correlates with intermediate and not high miR-155 expression identified in our experimental models. These findings collectively describe a novel dose-dependent role for miR-155 in the regulation of AML, which may have important therapeutic implications.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , MicroARNs/genética , Interferencia de ARN , Adolescente , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Niño , Preescolar , Modelos Animales de Enfermedad , Expresión Génica , Hematopoyesis/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Lactante , Recién Nacido , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Ratones , Pronóstico , Ensayo de Tumor de Célula MadreRESUMEN
Hoxb8 overexpression immortalises haematopoietic progenitor cells in a growth-factor-dependant manner and co-operates with interleukin-3 (IL-3) to cause acute myeloid leukaemia. To further understand how Hoxb8 contributes to myeloid cell immortalisation, we generated IL-3-dependant myeloid cells expressing Hoxb8 under the control of an inducible promoter. Downregulation of Hoxb8, in the presence of IL-3, caused cell-cycle arrest and apoptosis in the majority of cells. Apoptosis was dependant on Bax and Bak and, in part, on Bim, which was repressed by Hoxb8. Deletion of the miR-17â¼92 seed sequences in the Bim 3'UTR abolished Hoxb8-dependant regulation of Bim reporter constructs. Expression of all six miRNAs from this cluster were elevated when Hoxb8 was overexpressed. The miR-17â¼92 cluster was required for repression of Bim in Hoxb8-immortalised cells and deletion of the miR-17â¼92 cluster substantially inhibited Hoxb8, but not Hoxa9, mediated survival and proliferation. Hoxb8 appears to promote miR-17â¼92 expression through c-Myc, a known transcriptional regulator of the miR-17â¼92 cluster. We have uncovered a previously unrecognised link between Hoxb8 expression and microRNAs that provides a new insight into the oncogenic functions of Hoxb8.
Asunto(s)
Proteínas de Homeodominio/genética , MicroARNs/metabolismo , Regiones no Traducidas 3' , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Muerte Celular/genética , Diferenciación Celular/genética , Procesos de Crecimiento Celular/genética , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transfección , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The activation of the Akt signalling in response to cytokine receptor signalling promotes protein synthesis, cellular growth and proliferation. To determine the role of Akt in interleukin-3 (IL-3) signalling, we generated IL-3-dependent myeloid cell lines from mice lacking Akt1, Akt2 or Akt3. Akt1 deletion resulted in accelerated apoptosis at low concentrations of IL-3. Expression of constitutively active Akt1 was sufficient to delay apoptosis in response to IL-3 withdrawal, but not sufficient to induce proliferation in the absence of IL-3. Akt1 prolonged survival of Bim- or Bad-deficient cells, but not cells lacking Puma, indicating that Akt1-dependent repression of apoptosis was in part dependent on Puma and independent of Bim or Bad. Our data show that a key role of Akt1 during IL-3 signalling is to repress p53-dependent apoptosis pathways, including transcriptional upregulation of Puma. Moreover, our data indicate that regulation of BH3-only proteins by Akt is dispensable for Akt-dependent cell survival.
Asunto(s)
Apoptosis/fisiología , Citocinas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Procesos de Crecimiento Celular/fisiología , Células HEK293 , Humanos , Interleucina-3/metabolismo , Isoenzimas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/citología , Células Mieloides/enzimología , Receptores de Interleucina-3/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. METHODS: A method is described to massively expand bone marrow-derived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. RESULTS: IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). CONCLUSION: A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.
Asunto(s)
Basófilos/citología , Basófilos/fisiología , Diferenciación Celular/genética , Proteínas de Homeodominio/genética , Animales , Apoptosis/efectos de los fármacos , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Histamina/metabolismo , Interleucina-3/farmacología , Leucotrieno C4/metabolismo , Ratones , Células Th2/inmunología , Células Th2/metabolismo , Triptasas/genética , Triptasas/metabolismoRESUMEN
Cisplatin is a widely used cancer chemotherapeutic that promotes DNA damage-associated apoptosis. Although platinum compounds are known to form DNA adducts and provoke DNA damage, the molecular mechanism of cisplatin-induced cell death remains unclear. In this article, we show that the BH3-only protein Noxa is strongly transcriptionally upregulated in response to cisplatin and related platinum compounds. Cisplatin-induced Noxa expression was ERK dependent, but p53 independent, and inhibition of ERK activation markedly attenuated cisplatin-induced cell death, as well as Noxa expression. Furthermore, siRNA-mediated ablation of Noxa expression also inhibited cisplatin-induced cell death and permitted clonogenic survival. These observations reveal a novel ERK-regulated route to Noxa expression that is important for the cell killing activity of platinum-based chemotherapeutic drugs.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Cisplatino/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Compuestos de Platino/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Daño del ADN , Quinasas MAP Reguladas por Señal Extracelular/análisis , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Células HeLa , Humanos , Leupeptinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
CD95 (Fas/Apo-1)-mediated apoptosis was shown to occur through two distinct pathways. One involves a direct activation of caspase-3 by large amounts of caspase-8 generated at the DISC (Type I cells). The other is related to the cleavage of Bid by low concentration of caspase-8, leading to the release of cytochrome c from mitochondria and the activation of caspase-3 by the cytochrome c/APAF-1/caspase-9 apoptosome (Type II cells). It is also known that the protein synthesis inhibitor cycloheximide (CHX) sensitizes Type I cells to CD95-mediated apoptosis, but it remains contradictory whether this effect also occurs in Type II cells. Here, we show that sub-lethal doses of CHX render both Type I and Type II cells sensitive to the apoptogenic effect of anti-CD95 antibodies but not to chemotherapeutic drugs. Moreover, Bcl-2-positive Type II cells become strongly sensitive to CD95-mediated apoptosis by the addition of CHX to the cell culture. This is not the result of a restraint of the anti-apoptotic effect of Bcl-2 at the mitochondrial level since CHX-treated Type II cells still retain their resistance to chemotherapeutic drugs. Therefore, CHX treatment is granting the CD95-mediated pathway the ability to bypass the mitochondria requirement to apoptosis, much alike to what is observed in Type I cells.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Mitocondrias/metabolismo , Transducción de Señal/fisiología , Receptor fas/metabolismo , Anticuerpos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/efectos de los fármacos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Cicloheximida/farmacología , Citocromos c/efectos de los fármacos , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Sinergismo Farmacológico , Células HL-60 , Humanos , Mitocondrias/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor fas/antagonistas & inhibidoresAsunto(s)
Glicoproteínas de Membrana/fisiología , Neoplasias/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Glicoproteínas de Membrana/farmacología , Modelos Biológicos , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Apoptosis is an essential form of cell death, the failure of which can lead to cancer development. Cancer including leukemia is usually treated with chemotherapeutic drugs that can be effective, but frequently problems are encountered that impair the success of the treatment. Butyrate is a short-chain fatty acid that can have many effects on different cells, including apoptosis. METHODS: The effect of a combination treatment with butyrate and antineoplastic agents Ara-C, etoposide and vincristine is evaluate on the leukemic cell line THP-1. RESULTS: We show that butyrate increased apoptosis induced by the three agents as seen by measurement of DNA content, annexin exposure and morphological characteristics. We also demonstrate that the process of apoptosis induced by butyrate and chemotherapeutic drugs involves the participation of caspases and induced activation of caspase-3, -8 and -9. CONCLUSIONS: We believe that butyrate could be a promising therapeutic agent for the treatment of leukemia in combination with other antineoplastic drugs.
Asunto(s)
Antineoplásicos/farmacología , Butiratos/farmacología , Sinergismo Farmacológico , Leucemia Monocítica Aguda/tratamiento farmacológico , Leucemia Monocítica Aguda/patología , Clorometilcetonas de Aminoácidos/farmacología , Clorometilcetonas de Aminoácidos/uso terapéutico , Antineoplásicos/clasificación , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Western Blotting , Butiratos/química , Butiratos/uso terapéutico , Inhibidores de Caspasas , Caspasas/metabolismo , Caspasas/uso terapéutico , Línea Celular Tumoral , Citarabina/farmacología , Citarabina/uso terapéutico , Replicación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales/métodos , Quimioterapia Combinada , Etopósido/farmacología , Etopósido/uso terapéutico , Humanos , Leucemia Monocítica Aguda/metabolismo , Vincristina/farmacología , Vincristina/uso terapéuticoAsunto(s)
Apoptosis/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Timo/citología , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Ligando Inductor de Apoptosis Relacionado con TNFRESUMEN
Bcr-Abl is one of the most potent antiapoptotic molecules and is the tyrosine-kinase implicated in Philadelphia (Ph) chromosome-positive leukemia. It is still obscure how Bcr-Abl provides the leukemic cell a strong resistance to chemotherapeutic drugs. A rational drug development produced a specific inhibitor of the catalytic activity of Bcr-Abl called STI571. This drug was shown to eliminate Bcr-Abl-positive cells both in vitro and in vivo, although resistant cells may appear in culture and relapse occurs in some patients. In the study described here, Bcr-Abl-positive cells treated with tyrosine-kinase inhibitors such as herbimycin A, genistein or STI571 lost their phosphotyrosine-containing proteins, but were still extremely resistant to apoptosis. Therefore, in the absence of tyrosine-kinase activity, Bcr-Abl-positive cells continue to signal biochemically to prevent apoptosis induced by chemotherapeutic drugs. We propose that secondary antiapoptotic signals are entirely responsible for the resistance of Bcr-Abl-positive cells. Precise determination of such signals and rational drug development against them should improve the means to combat Ph chromosome-positive leukemia.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Piperazinas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/farmacología , Benzamidas , Benzoquinonas , Western Blotting , Caspasas/metabolismo , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Genisteína/farmacología , Células HL-60/efectos de los fármacos , Células HL-60/metabolismo , Humanos , Mesilato de Imatinib , Células K562/efectos de los fármacos , Células K562/metabolismo , Lactamas Macrocíclicas , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinonas/farmacología , Rifabutina/análogos & derivados , Factores de TiempoRESUMEN
Cyclobutane pyrimidine dimers (CPDs) are directly involved in signaling for UV-induced apoptosis in mammalian cells. Failure to remove these lesions, specially those located at actively expressing genes, is critical, as cells defective in transcription coupled repair have increased apoptotic levels. Thus, the blockage of RNA synthesis by lesions is an important candidate event triggering off active cell death. In this work, wild-type and XPB mutated Chinese hamster ovary (CHO) cells expressing a marsupial photolyase, that removes specifically CPDs from the damaged DNA, were generated, in order to investigate the importance of this lesion in both RNA transcription blockage and apoptotic induction. Photorepair strongly recovers RNA synthesis in wild-type CHO cell line, although the resumption of transcription is decreased in XPB deficient cells. This recovery is accompanied by the prevention of cells entering into apoptosis. These results demonstrate that marsupial photolyase has access to CPDs blocking RNA synthesis in vivo, and this may be affected by the presence of a mutated XPB protein.