RESUMEN
The transcription repressor BCL6 plays an essential role in the formation and function of germinal centers (GCs). While normal B cells promptly shut off BCL6 when they exit the GC, many GC-derived B-cell lymphomas sustain BCL6 expression through chromosomal translocations and activating mutations. We have previously shown that a common effect of lymphoma-associated BCL6 gene alterations is to bypass a negative autoregulatory loop that controls its transcription. In this study, we report that BCL6 autoregulation is independent of several known corepressor complexes including silencing mediator for retinoid and thyroid hormone receptors, nuclear receptor coreceptor, BCL6 corepressor, and MTA3/NuRD. Furthermore, we show that BCL6 can interact with the CtBP (C-terminal binding protein) corepressor both in vitro and in vivo and that CtBP is recruited by BCL6 to its 5' regulatory region. In lymphoma cell lines carrying BCL6 translocations, small interfering RNA-mediated CtBP knock-down selectively relieved the previously silenced wild-type BCL6 allele but not the translocated alleles, which are driven by heterologous promoters. These results demonstrate that CtBP is a novel BCL6 corepressor and suggest that a unique corepressor requirement for BCL6 autoregulation may allow GC B cells to differentially control the expression of BCL6 and other BCL6 target genes in response to environmental stimuli during the GC stage of B cell development.
Asunto(s)
Oxidorreductasas de Alcohol/fisiología , Linfocitos B/citología , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Represoras/fisiología , Oxidorreductasas de Alcohol/química , Línea Celular/metabolismo , Quimiocina CCL3/biosíntesis , Quimiocina CCL3/genética , Proteínas de Unión al ADN/química , Regulación de la Expresión Génica/genética , Humanos , Macrófagos/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/fisiología , Regiones Promotoras Genéticas/genética , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/fisiología , Proteínas Represoras/química , Proteínas Represoras/genéticaRESUMEN
In this study we evaluated UCN-01, a small molecule that inhibits protein kinases by interacting with the ATP-binding site, as a potential anti-cancer agent for neuroblastoma. UCN-01 was effective at inducing apoptosis in six neuroblastoma cell lines with diverse cellular and genetic phenotypes. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assays, detection of active caspase-3 and cleaved poly ADP-ribose polymerase (PARP) confirmed that UCN-01 induced apoptosis. Cell cycle analysis determined that the UCN-01 treated cells accumulated in S phase by 16 h. Unlike vinblastine and docetaxel that increased survivin expression, UCN-01 treatment did not increase X-linked inhibitor of apoptosis protein (XIAP) and survivin levels. Analysis of specific phosphoepitopes on chk1/2, Akt, and GSK3beta following UCN-01 treatment determined that there was no significant change in phospho-chk1/2. However, there was decreased immunoreactivity at Ser473 and Thr308 of Akt and Ser9 of GSK3beta by 4 h indicating that the Akt survival pathway and downstream signalling was compromised. Thus, UCN-01 was effective at inducing apoptosis in neuroblastoma cell lines.