Asunto(s)
Mieloma Múltiple , Osteólisis , Animales , Modelos Animales de Enfermedad , Genes myc , Humanos , Interleucina-6/genética , Ratones , Osteólisis/etiologíaRESUMEN
Prior work indicates that c-myc translation is up-regulated in multiple myeloma cells. To test a role for interleukin (IL)-6 in myc translation, we studied the IL-6-responsive ANBL-6 and IL-6-autocrine U266 cell lines as well as primary patient samples. IL-6 increased c-myc translation, which was resistant to rapamycin, indicating a mechanism independent of mammalian target of rapamycin (mTOR) and cap-dependent translation. In contrast, the cytokine enhanced cap-independent translation via a stimulatory effect on the myc internal ribosome entry site (IRES). As known IRES-trans-activating factors (ITAF) were unaffected by IL-6, we used a yeast-three-hybrid screen to identify novel ITAFs and identified hnRNP A1 (A1) as a mediator of the IL-6 effect. A1 specifically interacted with the myc IRES in filter binding assays as well as EMSAs. Treatment of myeloma cells with IL-6 induced serine phosphorylation of A1 and increased its binding to the myc IRES in vivo in myeloma cells. Primary patient samples also showed binding between A1 and the IRES. RNA interference to knock down hnRNP A1 prevented an IL-6 increase in myc protein expression, myc IRES activity, and cell growth. These data point to hnRNP A1 as a critical regulator of c-myc translation and a potential therapeutic target in multiple myeloma.
Asunto(s)
Genes myc , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Interleucina-6/farmacología , Mieloma Múltiple/genética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Ribosomas/metabolismo , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Ribosomas/genética , TransfecciónRESUMEN
Cellular interleukin 6 (IL-6) is an important growth factor for Kaposi sarcoma- associated herpesvirus (KSHV)-associated neoplasms, which include human immunodeficiency virus (HIV)-related and -unrelated cases of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Increased IL-6 levels are found in tissues affected with these diseases, and KSHV exists in a latent state in the majority of virally infected cells. In addition, acute infection with KSHV up-regulates IL-6 expression in endothelial cells. Thus, the hypothesis was considered that a latent KSHV gene product up-regulates IL-6 expression. To evaluate this hypothesis, the KSHV latency-associated nuclear antigen (LANA) was expressed in human embryonal kidney 293 cells and a bone marrow stromal cell line. LANA up-regulates IL-6 expression by inducing transcription from the IL-6 promoter, and the AP1 response element within the IL-6 promoter is necessary for and mediates IL-6 up-regulation by LANA. Thus, LANA may play a key pathophysiologic role in KSHV-associated neoplasms by functioning to up-regulate expression of IL-6.
Asunto(s)
Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/fisiología , Interleucina-6/biosíntesis , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Transcripción AP-1/metabolismo , Antígenos Virales , Línea Celular/metabolismo , Línea Celular/virología , Línea Celular Transformada , Humanos , Interleucina-6/genética , Riñón , FN-kappa B/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Eliminación de Secuencia , Células del Estroma/metabolismo , Transcripción Genética , Transfección , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Prior work demonstrates that AKT activity regulates sensitivity of cells to G(1) arrest induced by mammalian target of rapamycin (mTOR) inhibitors such as rapamycin and CCI-779. To investigate this, a novel high-throughput microarray polysome analysis was performed to identify genes whose mRNA translational efficiency was differentially affected following mTOR inhibition. The analysis also allowed the assessment of steady-state transcript levels. We identified two transcripts, cyclin D1 and c-myc, which exhibited differential expression in an AKT-dependent manner: High levels of activated AKT resulted in rapamycin-induced down-regulation of expression, whereas low levels resulted in up-regulation of expression. To ectopically express these proteins we exploited the finding that the p27(kip1) mRNA was efficiently translated in the face of mTOR inhibition irrespective of AKT activity. Thus, the p27(kip1) 5'-untranslated region was fused to the cyclin D1 and c-myc coding regions and these constructs were expressed in cells. In transfected cells, expression of cyclin D1 or c-myc was not decreased by rapamycin. Most importantly, this completely converted sensitive cells to a phenotype resistant to G(1) arrest. Furthermore, the AKT-dependent differential expression patterns of these two genes was also observed in a mouse xenograft model following in vivo treatment with CCI-779. These results identify two critical downstream molecular targets whose expression is regulated by AKT activity and whose down-regulation is required for rapamycin/CCI-779 sensitivity.