RESUMEN
BACKGROUND: Leukemia is a heterogeneous disease commonly associated with recurrent chromosomal translocations that involve tyrosine kinases including BCR-ABL, TEL-PDGFRB and TEL-JAK2. Most studies on the activated tyrosine kinases have focused on proximal signaling events, but little is known about gene transcription regulated by these fusions. METHODS: Oligonucleotide microarray was performed to compare mRNA changes attributable to BCR-ABL, TEL-PDGFRB and TEL-JAK2 after 1 week of activation of each fusion in Ba/F3 cell lines. Imatinib was used to control the activation of BCR-ABL and TEL-PDGFRB, and TEL-JAK2-mediated gene expression was examined 1 week after Ba/F3-TEL-JAK2 cells were switched to factor-independent conditions. RESULTS: Microarray analysis revealed between 800 to 2000 genes induced or suppressed by two-fold or greater by each tyrosine kinase, with a subset of these genes commonly induced or suppressed among the three fusions. Validation by Quantitative PCR confirmed that eight genes (Dok2, Mrvi1, Isg20, Id1, gp49b, Cxcl10, Scinderin, and collagen Vα1(Col5a1)) displayed an overlapping regulation among the three tested fusion proteins. Stat1 and Gbp1 were induced uniquely by TEL-PDGFRB. CONCLUSIONS: Our results suggest that BCR-ABL, TEL-PDGFRB and TEL-JAK2 regulate distinct and overlapping gene transcription profiles. Many of the genes identified are known to be involved in processes associated with leukemogenesis, including cell migration, proliferation and differentiation. This study offers the basis for further work that could lead to an understanding of the specificity of diseases caused by these three chromosomal translocations.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia/enzimología , Leucemia/genética , Proteínas Tirosina Quinasas/metabolismo , Translocación Genética , Benzamidas , Línea Celular Tumoral , Activación Enzimática , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas de Fusión Oncogénica/metabolismo , Piperazinas/farmacología , Reacción en Cadena de la Polimerasa/métodos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/genética , Pirimidinas/farmacología , ARN Mensajero/genéticaRESUMEN
The oncogene c-maf is frequently overexpressed in multiple myeloma cell lines and patient samples and contributes to increased cellular proliferation in part by inducing cyclin D2 expression. To identify regulators of c-maf, we developed a chemical screen in NIH3T3 cells stably overexpressing c-maf and the cyclin D2 promoter driving luciferase. From a screen of 2400 off-patent drugs and chemicals, we identified glucocorticoids as c-maf-dependent inhibitors of cyclin D2 transactivation. In multiple myeloma cell lines, glucocorticoids reduced levels of c-maf protein without influencing corresponding mRNA levels. Subsequent studies demonstrated that glucocorticoids increased ubiquitination-dependent degradation of c-maf and up-regulated ubiquitin C mRNA. Moreover, ectopic expression of ubiquitin C recapitulated the effects of glucocorticoids, demonstrating regulation of c-maf protein through the abundance of the ubiquitin substrate. Thus, using a chemical biology approach, we identified a novel mechanism of action of glucocorticoids and a novel mechanism by which levels of c-maf protein are regulated by the abundance of the ubiquitin substrate.
Asunto(s)
Glucocorticoides/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-maf/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Animales , Ciclina D2 , Ciclinas/genética , Ciclinas/metabolismo , Evaluación Preclínica de Medicamentos , Glucocorticoides/química , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Células 3T3 NIH , Regiones Promotoras Genéticas/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
Obesity is a well-defined risk factor for the development of insulin resistance in target tissues, such as skeletal muscle, and thus type 2 diabetes. This may occur due to endocrine effects mediated by adipokines including leptin, the product of the obese (ob) gene, whose circulating levels positively correlate with body mass index. Induction of suppressor of cytokine-3 (SOCS-3) protein expression has been implicated as a possible mechanism of leptin-induced insulin resistance. Here, we show that treatment of rat skeletal muscle cells with leptin activated the SOCS-3 gene promoter and caused a time-dependent increase in both SOCS-3 mRNA and protein content. Confocal microscopy demonstrated increased co-localization of SOCS-3 with insulin receptor in leptin-treated cells and we confirmed a direct interaction between these two proteins by showing increased coimmunoprecipitation of SOCS-3 and insulin receptor after exposure of cells to leptin. However, the expected functional consequences were not observed, as we saw no change in basal or insulin-stimulated glucose uptake and phosphorylation of GSK3beta, Akt (T308 and S473) or ERK1/2. In summary, leptin induced SOCS-3 expression and its association with the insulin receptor in rat skeletal muscle cells but functional significance of this increase was not apparent upon measuring glucose uptake.
