RESUMO
CTLA-4 and PD-1 are receptors that negatively regulate T-cell activation. Ligation of both CTLA-4 and PD-1 blocked CD3/CD28-mediated upregulation of glucose metabolism and Akt activity, but each accomplished this regulation using separate mechanisms. CTLA-4-mediated inhibition of Akt phosphorylation is sensitive to okadaic acid, providing direct evidence that PP2A plays a prominent role in mediating CTLA-4 suppression of T-cell activation. In contrast, PD-1 signaling inhibits Akt phosphorylation by preventing CD28-mediated activation of phosphatidylinositol 3-kinase (PI3K). The ability of PD-1 to suppress PI3K/AKT activation was dependent upon the immunoreceptor tyrosine-based switch motif located in its cytoplasmic tail, adding further importance to this domain in mediating PD-1 signal transduction. Lastly, PD-1 ligation is more effective in suppressing CD3/CD28-induced changes in the T-cell transcriptional profile, suggesting that differential regulation of PI3K activation by PD-1 and CTLA-4 ligation results in distinct cellular phenotypes. Together, these data suggest that CTLA-4 and PD-1 inhibit T-cell activation through distinct and potentially synergistic mechanisms.
Assuntos
Antígenos de Diferenciação/fisiologia , Antígenos de Superfície/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Ativação Linfocitária/fisiologia , Linfócitos T/fisiologia , Antígenos CD , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Antígeno CTLA-4 , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Ácido Okadáico/farmacologia , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Receptor de Morte Celular Programada 1 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
RNA interference is thought to require near-identity between the small interfering RNA (siRNA) and its cognate mRNA. Here, we used gene expression profiling to characterize the specificity of gene silencing by siRNAs in cultured human cells. Transcript profiles revealed siRNA-specific rather than target-specific signatures, including direct silencing of nontargeted genes containing as few as eleven contiguous nucleotides of identity to the siRNA. These results demonstrate that siRNAs may cross-react with targets of limited sequence similarity.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Marcação de Genes/métodos , Interferência de RNA , RNA Interferente Pequeno/genética , Sequência de Bases , Sequência Conservada , Inativação Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , RNA Mensageiro , Transformação GenéticaRESUMO
microRNAs in the miR-106b family are overexpressed in multiple tumor types and are correlated with the expression of genes that regulate the cell cycle. Consistent with these observations, miR-106b family gain of function promotes cell cycle progression, whereas loss of function reverses this phenotype. Microarray profiling uncovers multiple targets of the family, including the cyclin-dependent kinase inhibitor p21/CDKN1A. We show that p21 is a direct target of miR-106b and that its silencing plays a key role in miR-106b-induced cell cycle phenotypes. We also show that miR-106b overrides a doxorubicin-induced DNA damage checkpoint. Thus, miR-106b family members contribute to tumor cell proliferation in part by regulating cell cycle progression and by modulating checkpoint functions.
Assuntos
Ciclo Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Genes cdc , MicroRNAs/fisiologia , Proteínas de Neoplasias/fisiologia , Interferência de RNA , RNA Neoplásico/fisiologia , Mama/citologia , Linhagem Celular Transformada/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA , Doxorrubicina/toxicidade , Feminino , Humanos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genéticaRESUMO
High-capacity methods for assessing gene function have become increasingly important because of the increasing number of newly identified genes emerging from large-scale genome sequencing and cDNA cloning efforts. We investigated the use of DNA microarrays to identify uncharacterized genes specifically involved in human T cell activation. Activation of human peripheral blood T lymphocytes induced significant changes in hundreds of transcripts, but most of these were not unique to T cell activation. Variation of experimental parameters and analysis techniques allowed better enrichment for gene expression changes unique to T cell activation. Best results were achieved by identification of genes that were most highly coregulated with the T-cell-specific transcript interleukin 2 (IL2) in a "compendium" of experiments involving both T cells and other cell types. Among the genes most highly coregulated with IL2 were many genes known to function during T cell activation, together with ESTs of unknown function. Four of these ESTs were extended to novel full-length clones encoding T-cell-regulated proteins with predicted functions in GTP metabolism, cell organization, and signal transduction.