RESUMEN
Chronic lymphocytic leukemia (CLL) cells from clinically aggressive cases have a greater capacity to respond to external microenvironmental stimuli, including those transduced through Toll-like-receptor-9 (TLR9). Concomitant microRNA and gene expression profiling in purified CLL cells (n=17) expressing either unmutated (UM) or mutated (M) IGHV genes selected microRNAs from the miR-17â¼92 family as significantly upregulated and in part responsible for modifications in the gene expression profile of UM CLL cells stimulated with the TLR9 agonist CpG. Notably, the stable and sustained upregulation of miR-17â¼92 microRNAs by CpG was preceded by a transient induction of the proto-oncogene MYC. The enforced expression of miR-17, a major member from this family, reduced the expression of the tumor suppressor genes E2F5, TP53INP1, TRIM8 and ZBTB4, and protected cells from serum-free-induced apoptosis (P ≤ 0.05). Consistently, transfection with miR-17â¼92 family antagomiRs reduced Bromo-deoxy-uridine incorporation in CpG-stimulated UM CLL cells. Finally, miR-17 expression levels, evaluated in 83 CLL samples, were significantly higher in UM (P=0.03) and ZAP-70(high) (P=0.02) cases. Altogether, these data reveal a role for microRNAs of the miR-17â¼92 family in regulating pro-survival and growth-promoting responses of CLL cells to TLR9 triggering. Overall, targeting of this pathway may represent a novel therapeutic option for management of aggressive CLL.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , MicroARNs/genética , Mutación/genética , Receptor Toll-Like 9/genética , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Proliferación Celular , Perfilación de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Oligodesoxirribonucleótidos/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proto-Oncogenes Mas , ARN Largo no Codificante , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteína Tirosina Quinasa ZAP-70/genéticaRESUMEN
The protein kinase Syk is a key mediator of proximal B-cell receptor (BCR) signaling. Following antigen stimulation, Syk is recruited to the BCR and becomes activated by phosphorylation at Y352. Recently, Syk was found to be constitutively phosphorylated in several common B-cell lymphoma subtypes, indicating a role for antigen-independent Syk activation in the pathogenesis of these diseases. We now report that Syk is constitutively phosphorylated on the activating Y352 residue in chronic lymphocytic leukemia (CLL) B cells. To examine the effects of constitutive Syk activity on intracellular signaling and leukemic cell survival, we performed in vitro studies with the Syk inhibitor R406. Treatment with R406 induced leukemic cell apoptosis in the majority of investigated cases and affected the basal activity or expression of several pro-survival molecules regulated by Syk, including the Akt and extracellular signal-regulated (ERK) kinases, and the anti-apoptotic protein Mcl-1. In addition, R406 prevented the increase in leukemic cell viability induced by sustained BCR engagement and inhibited BCR-induced Akt activation and Mcl-1 upregulation. Collectively, these data identify Syk as a potential target for CLL treatment and suggest that inhibition of this kinase could provide a double therapeutic benefit by disrupting both antigen-dependent and antigen-independent signaling pathways that regulate leukemic cell survival.
Asunto(s)
Apoptosis/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis , Linfocitos B/patología , Supervivencia Celular , Regulación hacia Abajo/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana Edad , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Oxazinas/farmacología , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Piridinas/farmacología , Quinasa Syk , Células Tumorales CultivadasRESUMEN
Chronic lymphocytic leukemia (CLL) B-cells are hyporesponsive to many proliferative signals that induce activation of normal B-lymphocytes. However, a heterogeneous response has recently been observed with immunostimulatory CpG-oligodeoxynucleotides (CpG ODN). We now show that CpG ODN induce proliferation mainly in CLL B-cells from patients with progressive disease and unmutated immunoglobulin V(H) genes, whereas G(1)/S cell cycle arrest and apoptosis are induced in leukemic B-cells from stable/V(H) mutated CLL. Examination of early signaling events demonstrated that all CLL B-cells respond to CpG ODN stimulation by degradation of the NF-kappaB inhibitor IkappaB and activation of the Akt, ERK, JNK and p38 MAPK kinases, but the magnitude and duration of the signaling response was greater in the proliferating cases. Pharmacological inhibition of these pathways showed that simultaneous activation of Akt, ERK and JNK is required for cell cycle progression and proliferation. Conversely, introduction of constitutively active Akt in nonproliferating CLL B-cells resulted in induction of cyclin A following CpG ODN stimulation, indicating that increased Akt activation is sufficient to overcome the hyporesponsiveness of these cells to proliferative signals. Thus, the magnitude of Akt signaling may determine the distinct responses observed in leukemic B-cells belonging to the different prognostic subgroups.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Ciclo Celular , Proliferación Celular , Ciclinas/biosíntesis , Progresión de la Enfermedad , Femenino , Genes de Inmunoglobulinas , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Persona de Mediana Edad , Oligodesoxirribonucleótidos/inmunología , Oligodesoxirribonucleótidos/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
The Gas3/PMP22 protein family is characterized by tetraspan transmembrane proteins. The gas3/PMP22 gene is highly expressed in Schwann cells of the peripheral nervous system, and different alterations of this gene are associated with hereditary demyelinating neuropathies, such as the Charcot-Marie-Tooth type 1A, the Dejerine-Sottas syndrome and the Hereditary Liability to Pressure Palsies (HNPP).Here, we report on the identification of at least one member of the Gas3/PMP22 family in the nematode C. elegans (C01C10.1b). C01C10.1b shares 36% of identical amino acids with the human Gas3/PMP22 and is characterized by four hydrophobic putative transmembrane domains. It lacks the typical N-linked glycosylation consensus in the first extracellular loop. C01C10.1b is transcribed as an operon downstream to the gene C01C10.1a, which encodes for a putative tetraspan protein with less conserved homology with the Gas3/PMP22 family. Interestingly, C01C10.1a contains three N-glycosylation sites at the C-terminus. Both genes are expressed in different nematode developmental stages and in the adults. The characterization of one member of the gas3/PMP22 family in C. elegans gives the opportunity to use this model organism to investigate the role of gas3/PMP22 in the regulation of cell proliferation and differentiation and its relation to the hereditary neurodegenerative diseases in humans.