RESUMEN
BACKGROUND AND OBJECTIVES: Stem cells play an important role in the pathogenesis and maintenance of most malignant tumors. Acute myeloid leukemia (AML) is a stem cell disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARalpha fusion protein. Treatment of APL with all-trans retinoic acid (ATRA) induces CR, but not molecular remission (CMR), because the fusion transcript remains detectable, followed by relapse within a few months. Arsenic induces high rates of CR and CMR followed by a long relapse-free survival (RFS). Here we compared the effects of ATRA and arsenic on PML/RARalpha-positive stem cell compartments. DESIGN AND METHODS: As models for the PML/RARalpha-positive LSC we used: (i) Sca1+/lin- murine HSC retrovirally transduced with PML/RARalpha; (ii) LSC from mice with PML/RARalpha-positive leukemia; (iii) the side population of the APL cell line NB4. RESULTS: In contrast to ATRA, arsenic abolishes the aberrant stem cell capacity of PML/RARalpha-positive stem cells. Arsenic had no apparent influence on the proliferation of PML/RARalpha-positive stem cells, whereas ATRA greatly increased the proliferation of these cells. Furthermore ATRA induces proliferation of APL-derived stem cells, whereas arsenic inhibits their growth. INTERPRETATIONS AND CONCLUSIONS: Taken together our data suggest a relationship between the capacity of a compound to target the leukemia-initiating cell and its ability to induce long relapse-free survival. These data strongly support the importance of efficient LSC-targeting for the outcome of patients with leukemia.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Inhibidores de Crecimiento/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Leucemia Promielocítica Aguda/patología , Células Madre Neoplásicas/efectos de los fármacos , Óxidos/farmacología , Animales , Trióxido de Arsénico , Ataxina-1 , Ataxinas , Biomarcadores de Tumor/análisis , División Celular/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Femenino , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/genética , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/análisis , Proteínas Nucleares/análisis , Proteínas de Fusión Oncogénica/análisis , Proteínas de Fusión Oncogénica/fisiología , Proteínas Recombinantes de Fusión/fisiología , Transfección , Tretinoina/administración & dosificación , Tretinoina/farmacología , Ensayo de Tumor de Célula MadreRESUMEN
The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RARalpha), and PLZF-RARalpha encode aberrant transcription factors. Several lines of evidence suggest similar pathogenetic mechanisms for these fusion proteins. We used high-density oligonucleotide arrays to identify shared target genes in inducibly transfected U937 cells expressing AML1-ETO, PML-RARalpha, or PLZF-RARalpha. All three fusion proteins significantly repressed the expression of 38 genes and induced the expression of 14 genes. Several of the regulated genes were associated with Wnt signaling. One of these, plakoglobin (gamma-catenin), was induced on the mRNA and protein level by all three fusion proteins. In addition, primary AML blasts carrying one of the fusion proteins significantly overexpressed plakoglobin. The plakoglobin promoter was cloned and shown to be induced by AML1-ETO, with promoter activation depending on the corepressor and histone deacetylase binding domains. The induction of plakoglobin by AML fusion proteins led to downstream signaling and transactivation of TCF- and LEF-dependent promoters, including the c-myc promoter, which was found to be bound by plakoglobin in vivo after AML1-ETO expression. beta-Catenin protein levels and TCF and LEF target genes such as c-myc and cyclin D1 were found to be induced by the fusion proteins. On the functional level, a dominant negative TCF inhibited colony growth of AML1-ETO-positive Kasumi cells, whereas plakoglobin transfection into myeloid 32D cells enhanced proliferation and clonal growth. Injection of plakoglobin-expressing 32D cells into syngeneic mice accelerated the development of leukemia. Transduction of plakoglobin into primitive murine hematopoietic progenitor cells preserved the immature phenotype during colony growth, suggesting enhanced self-renewal. These data provide evidence that activation of Wnt signaling is a common feature of several balanced translocations in AML.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Células Madre Hematopoyéticas/fisiología , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra , Animales , Línea Celular Tumoral , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desmoplaquinas , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Factor de Unión 1 al Potenciador Linfoide , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Proteína 1 Compañera de Translocación de RUNX1 , Factores de Transcripción/genética , Trasplante Isogénico , Proteínas Wnt , gamma CateninaRESUMEN
Acute myeloid leukemia is characterized by a differentiation block as well as by an increased self-renewal of hematopoietic precursors in the bone marrow. This phenotype is induced by specific acute myeloid leukemia-associated translocations, such as t(15;17) and t(11;17), which involve an identical portion of the retinoic acid receptor alpha (RARalpha) and either the promyelocytic leukemia (PML) or promyelocytic zinc finger (PLZF) genes, respectively. The resulting fusion proteins form high molecular weight complexes and aberrantly bind several histone deacetylase-recruiting nuclear corepressor complexes. The amino-terminal BTB/POZ domain is indispensable for the capacity of PLZF to form high molecular weight complexes. Here, we studied the role of dimerization and binding to histone deacetylase-recruiting nuclear corepressor complexes for the induction of the leukemic phenotype by PLZF/RARalpha and we show that (a) the BTB/POZ domain mediates the oligomerization of PLZF/RARalpha; (b) mutations that inhibit dimerization of PLZF do the same in PLZF/RARalpha; (c) the PLZF/RARalpha-related block of differentiation requires an intact BTB/POZ domain; (d) the mutations interfering with either folding of the BTB/POZ domain or with its charged pocket prevent the self-renewal of PLZF/RARalpha-positive hematopoietic stem cells. Taken together, these data provide evidence that the dimerization capacity and the formation of a functionally charged pocket are indispensable for the PLZF/RARalpha-induced leukemogenesis.
Asunto(s)
Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/metabolismo , Enfermedad Aguda , Animales , Células COS , Dimerización , Femenino , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Mutación Puntual , Regiones Promotoras Genéticas , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Transcripción Genética , Dedos de ZincRESUMEN
The capacity of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) to preferentially induce apoptosis in malignant cells while sparing normal tissues renders it an attractive therapeutic agent. Nevertheless, the molecular determinants governing sensitivity towards TRAIL remain to be defined. Acknowledging the previously demonstrated deregulation of prostate-apoptosis-response-gene-4 (par-4) in ex vivo cells of patients suffering from acute and chronic lymphatic leukemia, we here tested the hypothesis that expression of par-4 influences sensitivity to TRAIL. Evaluating this hypothesis we show, that par-4-transfected T-lymphoblastic Jurkat cells exhibit a considerably increased rate of apoptosis upon incubation with an agonistic TRAIL-antibody as compared to their mock-transfected counterparts. Defining the underlying molecular mechanisms we provide evidence, that par-4 enhances sensitivity towards TRAIL by employing crucial members of the extrinsic pathway. Thus, par-4-overexpressing Jurkat clones show an enforced cleavage of c-Flip(L) together with an increased activation of the initiator caspases-8 and -10. In addition, expression of par-4 enables cells to down-regulate the inhibitor-of-apoptosis proteins cIAP-1, cIAP-2, XIAP and survivin with a concomitantly enhanced activation of the executioner caspases-6 and -7. Supporting the crucial role of caspase-8 in par-4-promoted apoptosis we demonstrate that inhibition of caspase-8 considerably reduces TRAIL-induced apoptosis in par-4 and mock-transfected Jurkat clones and reverses the described molecular changes. In conclusion, we here provide first evidence that expression of par-4 in neoplastic lymphocytes augments sensitivity to TRAIL-induced cell death and outline the responsible molecular mechanisms, in particular the crucial role of caspase-8 activation.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Glicoproteínas de Membrana/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/farmacología , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Caspasa 8 , Inhibidores de Caspasas , Colágeno Tipo XI/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis/efectos de los fármacos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat , Linfocitos/metabolismo , Linfocitos/patología , Glicoproteínas de Membrana/farmacología , Oligopéptidos/farmacología , Sensibilidad y Especificidad , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina-Proteína LigasasRESUMEN
BACKGROUND: The reciprocal (9;22) translocation fuses the bcr (breakpoint cluster region) gene on chromosome 22 to the abl (Abelson-leukemia-virus) gene on chromosome 9. Depending on the breakpoint on chromosome 22 (the Philadelphia chromosome--Ph+) the derivative 9+ encodes either the p40(ABL/BCR) fusion transcript, detectable in about 65% patients suffering from chronic myeloid leukemia, or the p96(ABL/BCR) fusion transcript, detectable in 100% of Ph+ acute lymphatic leukemia patients. The ABL/BCRs are N-terminally truncated BCR mutants. The fact that BCR contains Rho-GEF and Rac-GAP functions strongly suggest an important role in cytoskeleton modeling by regulating the activity of Rho-like GTPases, such as Rho, Rac and cdc42. We, therefore, compared the function of the ABL/BCR proteins with that of wild-type BCR. METHODS: We investigated the effects of BCR and ABL/BCRs i.) on the activation status of Rho, Rac and cdc42 in GTPase-activation assays; ii.) on the actin cytoskeleton by direct immunofluorescence; and iii) on cell motility by studying migration into a three-dimensional stroma spheroid model, adhesion on an endothelial cell layer under shear stress in a flow chamber model, and chemotaxis and endothelial transmigration in a transwell model with an SDF-1alpha gradient. RESULTS: Here we show that both ABL/BCRs lost fundamental functional features of BCR regarding the regulation of small Rho-like GTPases with negative consequences on cell motility, in particular on the capacity to adhere to endothelial cells. CONCLUSION: Our data presented here describe for the first time an analysis of the biological function of the reciprocal t(9;22) ABL/BCR fusion proteins in comparison to their physiological counterpart BCR.
Asunto(s)
Movimiento Celular/fisiología , Citoesqueleto/fisiología , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas Proto-Oncogénicas c-bcr/genética , Proteínas Proto-Oncogénicas c-bcr/fisiología , Humanos , Células Tumorales Cultivadas , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Acute myeloid leukemia (AML)-associated chromosomal translocations result in formation of chimeric transcription factors, such as PML/RARalpha, PLZF/RARalpha, and AML-1/ETO, of which the components are involved in regulation of transcription by chromatin modeling through histone acetylation/deacetylation. The leukemic differentiation block is attributed to deregulated transcription caused by these chimeric fusion proteins, which aberrantly recruit histone-deacetylase (HDAC) activity. One essential differentiation pathway blocked by the leukemic fusion proteins is the vitamin (Vit) D(3) signaling. Here we investigated the mechanisms by which the leukemic fusion proteins interfere with VitD(3)-induced differentiation. The VitD(3)-receptor (VDR) is, like the retinoid receptors RAR, retinoid X receptor, and the thyroid hormone receptor (TR), a ligand-inducible transcription factor. In the absence of ligand, the transcriptional activity of TR and RAR is silenced by recruitment of HDAC activity through binding to corepressors. In the presence of ligand, TR and RAR activate transcription by releasing HDAC activity and by recruiting histone-acetyltransferase activity. Here we report that VDR binds corepressors in a ligand-dependent manner and that inhibition of HDAC activity increases VitD(3) sensitivity of HL-60 cells. Nevertheless, the inhibition of HDAC activity is unable to overcome the block of VitD(3)-induced differentiation caused by PLZF/RARalpha expression. Here we demonstrate that the expression of the translocation products PML/RARalpha and PLZF/RARalpha impairs the localization of VDR in the nucleus by binding to VDR. Furthermore, the overexpression of VDR in U937 cells expressing AML-related translocation products completely abolishes the block of VitD(3)-induced differentiation. Taken together these data indicate that the AML-associated translocation products block differentiation not only by interfering with chromatin-modeling but also by sequestering factors involved in the differentiation signaling pathways, such as VDR in the VitD(3)-induced differentiation.
Asunto(s)
Colecalciferol/antagonistas & inhibidores , Leucemia Mieloide/patología , Proteínas de Fusión Oncogénica/fisiología , Receptores de Calcitriol/fisiología , Diferenciación Celular/fisiología , Colecalciferol/metabolismo , Colecalciferol/fisiología , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Células HL-60 , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Estructura Terciaria de Proteína , Proteína 1 Compañera de Translocación de RUNX1 , Receptores de Calcitriol/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Transfección , Translocación Genética , Tretinoina/farmacologíaRESUMEN
Inhibition of apoptosis is a hallmark of malignancies of the hematopoetic system. Previous studies in nonhematopoetic cells demonstrated that the prostate-apoptosis-response-gene-4 (Par-4) is up-regulated in cells undergoing programmed cell death and that Par-4 exerts its proapoptotic effect by down-regulating Bcl-2. After showing the aberrant expressional pattern of Par-4 in neoplastic lymphocytes as well as demonstrating inverse expressional patterns of Par-4 and Bcl-2 in malignant cells of patients suffering from acute lymphocytic leukemia, we assessed the functional consequences of Par-4 overexpression during apoptosis in Jurkat T lymphocytes. We show that in lymphatic cells Par-4 overexpression decreases the level of Bcl-2, whereas Bax, the proapoptotic counterpart of Bcl-2, retains unaltered levels. Moreover, Par-4 overexpression is accompanied by cleavage of poly(ADP-ribose) polymerase (PARP). Despite these effects, overexpression of Par-4 alone is not sufficient to induce apoptosis but markedly increases the rate of apoptosis on treatment with different chemotherapeutic agents. On chemotherapeutic treatment Par-4 overexpression enhances disruption of mitochondrial membrane potential, PARP-cleaving activity, as well as activation of caspase-3. The hypothesis of caspase-dependency of Par-4-promoted apoptosis is additionally supported by demonstrating complete abrogation of programmed cell death after pretreatment with a broad spectrum caspase-inhibitor. On inhibition of caspase-3 overexpression of Par-4 enables lymphatic cells to alternatively activate caspases-9, -6, and -7 by diminishing the influence of the inhibitors of apoptosis proteins (IAPs) cIAP1 and XIAP. Our study is the first to identify Par-4 as a proapoptotic protein in lymphatic cells, outlining a model of action evaluating the role of Bcl-2/Bax, as well as demonstrating the impact of Par-4 expression on PARP cleavage, disruption of mitochondrial membrane potential, caspase activation, and interactions with inhibitors of apoptosis proteins.
Asunto(s)
Apoptosis/fisiología , Proteínas Portadoras/fisiología , Caspasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Mitocondrias/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Inhibidores de Caspasas , Citarabina/farmacología , Regulación hacia Abajo , Doxorrubicina/farmacología , Activación Enzimática , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Células Jurkat/citología , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mitocondrias/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , TransfecciónRESUMEN
Since the 19th century, arsenic (As2O3) has been used in the treatment of chronic myelogenous leukemia (CML) characterized by the t(9;22) translocation. As2O3 induces complete remissions in patients with acute promyelocytic leukemia. The response to As2O3 is genetically determined by the t(15;17)-or the t(9;22)-specific fusion proteins PML/RARalpha or BCR/ABL. The PML portion of PML/RARalpha is crucial for the sensitivity to As2O3. PML is nearly entirely contained in PML/RARalpha. PML is upregulated by oncogenic RAS in primary fibroblasts. The aberrant kinase activity of BCR/ABL leads to constitutive activation of RAS. Therefore, we hypothesized that BCR/ABL could increase sensitivity to As2O2-induced apoptosis by modifying PML expression. To disclose the mechanism of As2O3-induced apoptosis in PML/RARalpha- and BCR/ABL-expressing cells, we focused on the role of PML for As2O3-induced cell death. Here we report that (i) sensitivity to As2O3-induced apoptosis of U937 cells can be increased either by overexpression of PML, or by conditional expression of activated RAS; (ii) also the expression of the t(8;21)-related AML-1/ETO increased sensitivity to As2O3-induced apoptosis; (iii) both BCR/ABL and AML-1/ETO activated RAS and modified the PML expression pattern; (iv) the expression of either BCR/ABL or AML-1/ETO rendered U937 cells sensitive to interferon alpha-induced apoptosis. In summary, these data suggest a crucial role of factors able to upregulate PML for As2O2-induced cell death.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Proteínas de Fusión bcr-abl/metabolismo , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Genes ras , Leucemia Promielocítica Aguda/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Apoptosis/efectos de los fármacos , Arsenicales/uso terapéutico , Benzamidas , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/genética , Monocitos/efectos de los fármacos , Óxidos/farmacología , Óxidos/uso terapéutico , Cromosoma Filadelfia , Piperazinas/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-abl/genética , Pirimidinas/farmacología , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Transgenes , Translocación Genética , Células Tumorales Cultivadas , Células U937 , Regulación hacia ArribaRESUMEN
OBJECTIVE: Prostate apoptosis response gene-4 (par-4) is deregulated in acute and chronic lymphatic leukemia. Given its pro-apoptotic role in neoplastic lymphocytes and evidence that par-4 antagonizes oncogenic Ras in solid tumors, we hypothesized that par-4 may act as a tumor suppressor impairing transformation induced by p185(BCR-ABL). MATERIALS AND METHODS: The capacity of par-4 to interfere with factor independence induced by p185(BCR-ABL) and V12ras was evaluated by analysis of factor-independent growth of p185(BCR-ABL)/ par-4 and V12ras/par-4 transduced cells. The expression of par-4 and p185(BCR-ABL) by the respective constructs was controlled by Western blot analysis. Activated Ras was detected by pull-down assay in the cell clones expressing p185(BCR-ABL) in the absence and presence of par-4. RESULTS: Expression of p185(BCR-ABL) causes factor independence, signifying a conversion toward a transformed phenotype in hematopoietic precursors. We demonstrate that par-4 completely abolishes factor independence induced by p185(BCR-ABL) and partially abrogates factor independence caused by activated V12ras. Evaluating the underlying molecular mechanisms, we show that par-4 hinders activation of oncogenic Ras and causes concomitant disruptions of p185(BCR-ABL)-mediated signaling. CONCLUSION: We provide the first evidence that par-4 exhibits an antitransforming capacity by antagonizing p185(BCR-ABL)-induced factor-independent proliferation in hematopoietic cells.
Asunto(s)
Apoptosis/genética , Proteínas Portadoras/genética , Células Madre Hematopoyéticas/citología , Péptidos y Proteínas de Señalización Intracelular , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , División Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular/genética , Clonación Molecular , Ensayo de Unidades Formadoras de Colonias , Proteínas de Fusión bcr-abl/genética , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Humanos , Interleucina-3/antagonistas & inhibidores , Interleucina-3/farmacología , Leucemia Linfocítica Crónica de Células B/genética , Masculino , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Próstata , Ratas , TransfecciónRESUMEN
Nonsteroidal anti-inflammatory drugs such as sulindac inhibit Wnt signaling, which is critical to maintain cancer stem cell-like cells (CSC), but they also suppress the activity of 5-lipoxygenase (5-LO) at clinically feasible concentrations. Recently, 5-LO was shown to be critical to maintain CSC in a model of chronic myeloid leukemia. For these reasons, we hypothesized that 5-LO may offer a therapeutic target to improve the management of acute myeloid leukemia (AML), an aggressive disease driven by CSCs. Pharmacologic and genetic approaches were used to evaluate the effects of 5-LO blockade in a PML/RARα-positive model of AML. As CSC models, we used Sca-1(+)/lin(-) murine hematopoietic stem and progenitor cells (HSPC), which were retrovirally transduced with PML/RARα. We found that pharmacologic inhibition of 5-LO interfered strongly with the aberrant stem cell capacity of PML/RARα-expressing HSPCs. Through small-molecule inhibitor studies and genetic disruption of 5-LO, we also found that Wnt and CSC inhibition is mediated by the enzymatically inactive form of 5-LO, which hinders nuclear translocation of ß-catenin. Overall, our findings revealed that 5-LO inhibitors also inhibit Wnt signaling, not due to the interruption of 5-LO-mediated lipid signaling but rather due to the generation of a catalytically inactive form of 5-LO, which assumes a new function. Given the evidence that CSCs mediate AML relapse after remission, eradication of CSCs in this setting by 5-LO inhibition may offer a new clinical approach for immediate evaluation in patients with AML. Cancer Res; 74(18); 5244-55. ©2014 AACR.
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Araquidonato 5-Lipooxigenasa/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Inhibidores de la Lipooxigenasa/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Animales , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Leucemia Mieloide Aguda/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Plásmidos , Transducción de Señal , TransfecciónRESUMEN
In acute promyelocytic leukemia (APL), hematopoietic differentiation is blocked and immature blasts accumulate in the bone marrow and blood. APL is associated with chromosomal aberrations, including t(15;17) and t(11;17). For these two translocations, the retinoic acid receptor alpha (RARα) is fused to the promyelocytic leukemia (PML) gene or the promyelocytic zinc finger (PLZF) gene, respectively. Both fusion proteins lead to the formation of a high-molecular-weight complex. High-molecular-weight complexes are caused by the "coiled-coil" domain of PML or the BTB/POZ domain of PLZF. PML/RARα without the "coiled-coil" fails to block differentiation and mediates an all-trans retinoic acid-response. Similarly, mutations in the BTB/POZ domain disrupt the high-molecular-weight complex, abolishing the leukemic potential of PLZF/RARα. Specific interfering polypeptides were used to target the oligomerization domain of PML/RARα or PLZF/RARα. PML/RARα and PLZF/RARα were analyzed for the ability to form high-molecular-weight complexes, the protein stability and the potential to induce a leukemic phenotype in the presence of the interfering peptides. Expression of these interfering peptides resulted in a reduced replating efficiency and overcame the differentiation block induced by PML/RARα and PLZF/RARα in murine hematopoietic stem cells. This expression also destabilized the PLZF/RARα-induced high-molecular-weight complex formation and caused the degradation of the fusion protein. Targeting fusion proteins through interfering peptides is a promising approach to further elucidate the biology of leukemia.
Asunto(s)
Leucemia Promielocítica Aguda/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Péptidos/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Promielocítica Aguda/genética , Ratones , Peso Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Péptidos/química , Péptidos/genética , Unión Proteica , Multimerización de Proteína , Proteolisis , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismoRESUMEN
AML progenitor cells (AML-PC) undergo significant apoptosis in response to the deacetylase inhibitor (DACi) LAQ824 and lose the replating capacity which was not observed with the DACi valproic acid. Treatment of normal hematopoietic progenitor cells (HPC) with LAQ824 resulted in (i) inhibition of differentiation, (ii) an G2/M cell cycle arrest exclusively in multipotent CD34(+) HPC and (iii) induction of apoptosis predominantly in committed CD34(-) HPC. Gene expression analysis showed induction of coactivator and target genes of the notch pathway as well as cell cycle arrest-inducing genes in the most primitive CD34(+) CD38(-) HPC population which may in part be responsible for the considerable, but reversible haematotoxicity of this drug.
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Células Madre Hematopoyéticas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , HumanosRESUMEN
Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and ß-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both ß-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Proteínas de Fusión Oncogénica/metabolismo , Sulindac/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Ratones , Fenotipo , Transducción de Señal/efectos de los fármacos , Sulindac/farmacología , Factores de Tiempo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , gamma Catenina/metabolismoRESUMEN
Translocations involving the abl locus on chromosome 9 fuses the tyrosine kinase c-ABL to proteins harboring oligomerization interfaces such as BCR or TEL, enabling these ABL-fusion proteins (X-ABL) to transform cells and to induce leukemia. The ABL kinase activity is blocked by the ABL kinase inhibitor STI571 which abrogates transformation by X-ABL. To investigate the role of oligomerization for the transformation potential of X-ABL and for the sensitivity to STI571, we constructed ABL chimeras with oligomerization interfaces of proteins involved in leukemia-associated translocations such as BCR, TEL, PML, and PLZF. We assessed the capacity of these chimeras to form high molecular weight (HMW) complexes as compared with p185(BCR-ABL). There was a direct relationship between the size of HMW complexes formed by these chimeras and their capacity to induce factor independence in Ba/F3 cells, whereas there was an inverse relationship between the size of the HMW complexes and the sensitivity to STI571. The targeting of the oligomerization interface of p185(BCR-ABL) by a peptide representing the coiled coil region of BCR reduced its potential to transform fibroblasts and increased sensitivity to STI571. Our results indicate that targeting of the oligomerization interfaces of the X-ABL enhances the effects of STI571 in the treatment of leukemia caused by X-ABL.
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Proteínas de Fusión bcr-abl/química , Proteínas Oncogénicas/metabolismo , Piperazinas/farmacología , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas , Pirimidinas/farmacología , Animales , Antineoplásicos/farmacología , Benzamidas , Western Blotting , Células COS , Línea Celular , Línea Celular Transformada , Transformación Celular Neoplásica , ADN Complementario/metabolismo , Fibroblastos/metabolismo , Mesilato de Imatinib , Concentración 50 Inhibidora , Leucemia/tratamiento farmacológico , Ratones , Microscopía Fluorescente , Mutación , Fosforilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcr , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Retroviridae/genética , Factores de TiempoRESUMEN
OBJECTIVE: The anti-inflammatory action of low-dose methoxetrate (MTX) in the treatment of rheumatoid arthritis (RA) appears to be partially impaired by folate supplementation. Here we investigated whether a folate excess impairs monocyte differentiation, a putative anti-inflammatory action of low-dose MTX. METHODS: Monocyte differentiation of U937 promonocytic cells was assessed by CD11b and CD14 immunostaining and fluorescent absorbent cell sorting (FACS) analysis. Cell proliferation and viability were determined by cell counts and trypan-blue staining, respectively. Nuclear apoptosis was assessed by 7-actinomycin staining. Cells were treated with 10(-10)-10(-6) M MTX in the presence or absence of folinic acid. Exposure to 1,25-OH-vitamine D(3) and TGF-beta served as a positive control of monocyte differentiation in U937 cells. RESULTS: Low-dose MTX-induced monocyte differentiation was marginal when compared with 1,25-OH-D(3) + TGF-beta treatment. Low-dose MTX inhibited cell proliferation, induced apoptosis, and reduced cell viability. All the antiproliferative, cytotoxic, and monocyte differentiating effects of MTX were completely reversed by folinic acid. CONCLUSIONS: Monocyte differentiation is part of the folate-dependent MTX actions.
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Antirreumáticos/farmacología , Leucovorina/farmacología , Metotrexato/farmacología , Monocitos/efectos de los fármacos , Células Madre/efectos de los fármacos , Apoptosis , Antígenos CD11/metabolismo , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Antagonismo de Drogas , Citometría de Flujo , Humanos , Receptores de Lipopolisacáridos/metabolismo , Monocitos/metabolismo , Monocitos/patología , Células Madre/metabolismo , Células Madre/patología , Células Tumorales CultivadasRESUMEN
Par-4 functions as a tumor suppressor antagonizing the transforming capacity and the resistance of malignant cells towards apoptotic stimuli. After demonstrating that par-4 promotes apoptosis by activating signaling of the intrinsic pathway of apoptosis, we hypothesized that par-4 also impacts on key molecules of the extrinsic pathway without the requirement of a receptor/ligand interaction. Here, we provide first evidence, that expression of par-4 increases cleavage of caspase-8, truncation of Bid and its translocation to the mitochondria, resulting in an augmentation of cytochrome c and AIF efflux into the cytosol, effects par-4-positive cells are able to retain to a higher extent than par-4-negative cells upon inhibition of caspase-3 activation.
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Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Citocromos c/metabolismo , Doxorrubicina/farmacología , Mitocondrias/metabolismo , Proteínas Reguladoras de la Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/metabolismo , Caspasa 8 , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Células Jurkat , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Acute myeloid leukemia (AML) is characterized by the block of differentiation, deregulated apoptosis, and an increased self-renewal of hematopoietic precursors. It is unclear whether the self-renewal of leukemic blasts results from the cumulative effects of blocked differentiation and impaired apoptosis or whether there are mechanisms directly increasing self-renewal. The AML-associated translocation products (AATPs) promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha), promyelocytic leukemia zinc finger (PLZF)/RAR alpha (X-RAR alpha), and AML-1/ETO block hematopoietic differentiation. The AATPs activate the Wnt signaling by up-regulating gamma-catenin. Activation of the Wnt signaling augments self-renewal of hematopoietic stem cells (HSCs). Therefore, we investigated how AATPs influence self-renewal of HSCs and evaluated the role of gamma-catenin in the determination of the phenotype of HSCs expressing AATPs. Here we show that the AATPs directly activate the gamma-catenin promoter. The crucial role of gamma-catenin in increasing the self-renewal of HSCs upon expression of AATPs is demonstrated by (i) the abrogation of replating efficiency upon hindrance of gamma-catenin expression through RNA interference, and (ii) the augmentation of replating efficiency of HSCs upon overexpression of gamma-catenin itself. In addition, the inoculation of gamma-catenin-transduced HSCs into irradiated recipient mice establishes the clinical picture of AML. These data provide the first evidence that the aberrant activation of Wnt signaling by the AATP decisively contributes to the pathogenesis of AML.