RESUMEN
Glioma is the most common adult primary brain tumor. Its most malignant form, glioblastoma multiforme (GBM), is almost invariably fatal, due in part to the intrinsic resistance of GBM to radiation- and chemotherapy-induced apoptosis. We analyzed B-cell leukemia-2 (Bcl-2) anti-apoptotic proteins in GBM and found myeloid cell leukemia-1 (Mcl-1) to be the highest expressed in the majority of malignant gliomas. Mcl-1 was functionally important, as neutralization of Mcl-1 induced apoptosis and increased chemotherapy-induced apoptosis. To determine how Mcl-1 was regulated in glioma, we analyzed the promoter and identified a novel functional single nucleotide polymorphism in an uncharacterized E26 transformation-specific (ETS) binding site. We identified the ETS transcription factor ELK4 as a critical regulator of Mcl-1 in glioma, since ELK4 downregulation was shown to reduce Mcl-1 and increase sensitivity to apoptosis. Importantly the presence of the single nucleotide polymorphism, which ablated ELK4 binding in gliomas, was associated with lower Mcl-1 levels and a greater dependence on Bcl-xL. Furthermore, in vivo, ELK4 downregulation reduced tumor formation in glioblastoma xenograft models. The critical role of ELK4 in Mcl-1 expression and protection from apoptosis in glioma defines ELK4 as a novel potential therapeutic target for GBM.
Asunto(s)
Apoptosis , Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , Glioblastoma/prevención & control , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína Elk-4 del Dominio ets/metabolismo , Adulto , Animales , Secuencia de Bases , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/prevención & control , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Regulación hacia Abajo , Ensayo de Cambio de Movilidad Electroforética , Glioblastoma/metabolismo , Humanos , Luciferasas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Datos de Secuencia Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Clasificación del Tumor , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Proteína Elk-4 del Dominio ets/antagonistas & inhibidores , Proteína Elk-4 del Dominio ets/genéticaRESUMEN
The Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción GATA/metabolismo , Leucemia/metabolismo , Metaloproteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Telomerasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Factores de Transcripción GATA/genética , Regulación del Desarrollo de la Expresión Génica , Genoma/genética , Proteínas con Dominio LIM , Leucemia/genética , Metaloproteínas/genética , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Telomerasa/genética , Análisis de Matrices Tisulares , Transactivadores/genéticaRESUMEN
Eph receptor tyrosine kinases (RTKs) are a highly conserved family of signaling proteins with functions in cellular migration, adhesion, apoptosis, and proliferation during both adult and embryonic life. Here, we describe a knock-in mouse in which EphA1 expression is disrupted via the insertion of an internal ribosome entry site (IRES)-human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene. This was shown to successfully knockout expression of endogenous EphA1 and enforce expression of the ALPP reporter by the EphA1 promoter. Staining for the ALPP reporter protein demonstrated an epithelially restricted expression pattern in mouse tissues. In EphA1 null mice, two separate phenotypes were identified: abnormal tail development manifesting as a kinky tail was found in approximately 80% of homozygous adults. A second, distinct abnormality present in approximately 18% of females was characterized by imperforate uterovaginal development with hydrometrocolpos and caused by a resistance of cells to apoptosis during reproductive tract canalization. These results indicate a possible role for EphA1 in tissue patterning and hormone-induced apoptotic processes.