RESUMO
Mutations in the Treacher Collins syndrome gene, TCOF1, cause a disorder of craniofacial development. We manipulated the levels of Tcof1 and its protein treacle in a murine neuroblastoma cell line to identify downstream changes in gene expression using a microarray platform. We identified a set of genes that have similar expression with Tcof1 as well as a set of genes that are negatively correlated with Tcof1 expression. We also showed that the level of Tcof1 and treacle expression is downregulated during differentiation of neuroblastoma cells into neuronal cells. Inhibition of Tcof1 expression by siRNA induced morphological changes in neuroblastoma cells that mimic differentiation. Thus, expression of Tcof1 and treacle synthesis play an important role in the proliferation of neuroblastoma cells and we have identified genes that may be important in this pathway.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Animais , Linhagem Celular Tumoral , Peptídeos e Proteínas de Sinalização Intracelular , Disostose Mandibulofacial/genética , Camundongos , Neuroblastoma/genética , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Megakaryocytic and erythroid lineages derive from a common bipotential progenitor and share many transcription factors, most prominently factors of the GATA zinc-finger family. Little is known about transcription factors unique to the megakaryocytic lineage that might program divergence from the erythroid pathway. To identify such factors, we used the K562 system in which megakaryocyte lineage commitment is dependent on sustained extracellular regulatory kinase (ERK) activation and is inhibited by stromal cell contact. During megakaryocytic induction in this system, the myeloid transcription factor RUNX1 underwent up-regulation, dependent on ERK signaling and inhibitable by stromal cell contact. Immunostaining of healthy human bone marrow confirmed a strong expression of RUNX1 and its cofactor, core-binding factor beta (CBFbeta), in megakaryocytes and a minimal expression in erythroblasts. In primary human hematopoietic progenitor cultures, RUNX1 and CBFbeta up-regulation preceded megakaryocytic differentiation, and down-regulation of these factors preceded erythroid differentiation. Functional studies showed cooperation among RUNX1, CBFbeta, and GATA-1 in the activation of a megakaryocytic promoter. By contrast, the RUNX1-ETO leukemic fusion protein potently repressed GATA-1-mediated transactivation. These functional interactions correlated with physical interactions observed between GATA-1 and RUNX1 factors. Enforced RUNX1 expression in K562 cells enhanced the induction of the megakaryocytic integrin proteins alphaIIb and alpha2. These results suggest that RUNX1 may participate in the programming of megakaryocytic lineage commitment through functional and physical interactions with GATA transcription factors. By contrast, RUNX1-ETO inhibition of GATA function may constitute a potential mechanism for the blockade of erythroid and megakaryocytic differentiation seen in leukemias with t(8;21).
Assuntos
Proteínas de Ligação a DNA/fisiologia , Megacariócitos/citologia , Proteínas Proto-Oncogênicas , Fatores de Transcrição/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/biossíntese , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Regulação da Expressão Gênica , Humanos , Integrina alfa2/análise , Células K562 , Leucemia/etiologia , Megacariócitos/química , Proteínas de Fusão Oncogênica/fisiologia , Glicoproteína IIb da Membrana de Plaquetas/análise , Fator de Transcrição AP-2 , Fatores de Transcrição/biossínteseRESUMO
Coculture with stromal cells tends to maintain normal hematopoietic progenitors and their leukemic counterparts in an undifferentiated, proliferative state. An example of this effect is seen with megakaryocytic differentiation, wherein stromal contact renders many cell types refractory to potent induction stimuli. This inhibitory effect of stroma on megakaryocytic differentiation correlates with a blockade within hematopoietic cells of protein kinase C-epsilon (PKC-epsilon) up-regulation and of extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase activation, both of which have been implicated in promoting megakaryocytic differentiation. In this study K562DeltaRafER.5 cells, expressing an estradiol-responsive mutant of the protein kinase Raf-1, were used to determine the relevance and stage of ERK/MAPK pathway blockade by stromal contact. Activation of DeltaRafER by estradiol overrode stromal blockade of megakaryocytic differentiation, implicating the proximal stage of the ERK/MAPK pathway as a relevant control point. Because stromal contact blocked delayed but not early ERK activation, the small guanosine triphosphatase (GTPase) Rap1 was considered as a candidate inhibitory target. Activation assays confirmed that Rap1 underwent sustained activation as a result of megakaryocytic induction, as previously described. As with ERK activation, stromal contact selectively blocked delayed but not early Rap1 activation, having no effect on Ras activation. Enforced expression of either wild-type Rap1 or the GTPase (GAP) resistant mutant Rap1 V12 failed to override stromal inhibition, suggesting that the inhibitory mechanism does not involve GAP up-regulation but rather may target upstream guanine nucleotide exchange factor (GEF) complexes. Accordingly, coimmunoprecipitation demonstrated stromally induced alterations in a protein complex associated with c-Cbl, a scaffolding factor for Rap1-GEF complexes.