RESUMO
Interleukin (IL)-7 is essential for normal T cell development. Previously, we have shown that IL-7 increases viability and proliferation of T cell acute lymphoblastic leukemia (T-ALL) cells by up-regulating Bcl-2 and down-regulating the cyclin-dependent kinase inhibitor p27kip1. Here, we examined the signaling pathways via which IL-7 mediates these effects. We investigated mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (Erk) and phosphatidylinositol-3-kinase (PI3K)-Akt (protein kinase B) pathways, which have active roles in T cell expansion and have been implicated in tumorigenesis. IL-7 induced activation of the MEK-Erk pathway in T-ALL cells; however, inhibition of the MEK-Erk pathway by the use of the cell-permeable inhibitor PD98059, did not affect IL-7-mediated viability or cell cycle progression of leukemic cells. IL-7 induced PI3K-dependent phosphorylation of Akt and its downstream targets GSK-3, FOXO1, and FOXO3a. PI3K activation was mandatory for IL-7-mediated Bcl-2 up-regulation, p27kip1 down-regulation, Rb hyperphosphorylation, and consequent viability and cell cycle progression of T-ALL cells. PI3K signaling was also required for cell size increase, up-regulation of CD71, expression of the glucose transporter Glut1, uptake of glucose, and maintenance of mitochondrial integrity. Our results implicate PI3K as a major effector of IL-7-induced viability, metabolic activation, growth and proliferation of T-ALL cells, and suggest that PI3K and its downstream effectors may represent molecular targets for therapeutic intervention in T-ALL.
Assuntos
Glucose/metabolismo , Interleucina-7/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Linfócitos T/metabolismo , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Sobrevivência Celular , Inibidor de Quinase Dependente de Ciclina p27 , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Transportador de Glucose Tipo 1 , Humanos , Immunoblotting , Imunofenotipagem , Lectinas Tipo C , Potenciais da Membrana , Mitocôndrias/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Fosforilação , Testes de Precipitina , Receptores da Transferrina , Transdução de Sinais , Fatores de Tempo , Proteínas Supressoras de Tumor/metabolismo , Regulação para CimaRESUMO
Adenoviral (Ad) transduction of dendritic cells (DC) is a promising vaccination strategy. However, clinical applicability of Ad vectors is hampered by the necessity to use high titers of infectious Ad particles for efficient DC transduction. Here, we report on the production of a bacterially expressed bispecific conjugate, consisting of a fusion of recombinant single-chain (sc) mAb Fv fragments, which bind and neutralize the Ad fiber knob (through the S11 mAb scFv) and retarget Ad to CD40 on the DC surface (through the G28-5 mAb scFv). We show that this bispecific scFv fusion protein significantly enhances transduction efficiency of monocyte-derived DC (MoDC), reduces the amount of virus needed for a given level of transduction, and increases the ability of MoDC to activate CTL in an antigen specific manner. This single-component conjugate may prove to be a valuable immunotherapeutic tool for the targeting of Ad to DC in vivo.