RESUMO
Notch1 activating mutations occur in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases and increase expression of Notch1 target genes, some of which activate AKT. HES1 transcriptionally silences phosphatase and tensin homolog (PTEN), resulting in AKT activation, which is reversed by Notch1 inhibition with γ-secretase inhibitors (GSIs). Mutational loss of PTEN is frequent in T-ALL and promotes resistance to GSIs due to AKT activation. GSI treatments increased AKT-Thr(308) phosphorylation and signaling in PTEN-deficient, GSI-resistant T-ALL cell lines (Jurkat, CCRF-CEM, and MOLT3), suggesting that Notch1 represses AKT independent of its PTEN transcriptional effects. AKT-Thr(308) phosphorylation and downstream signaling were also increased by knocking down Notch1 in Jurkat (N1KD) cells. This was blocked by treatment with the AKT inhibitor perifosine. The PI3K inhibitor wortmannin and the protein phosphatase type 2A (PP2A) inhibitor okadaic acid both impacted AKT-Thr(308) phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr(308) by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr(172) and p70S6K-Thr(389), both PP2A substrates, were also increased in both N1KD and GSI-treated cells and responded to okadaic acid treatment. A transcriptional regulatory mechanism was implied because ectopic expression of dominant-negative mastermind-like protein 1 increased and wild-type HES1 decreased phosphorylation of these PP2A targets. This was independent of changes in PP2A subunit levels or in vitro PP2A activity, but was accompanied by decreased association of PP2A with AKT in N1KD cells. These results suggest that Notch1 can regulate PP2A dephosphorylation of critical cellular regulators including AKT, AMPKα, and p70S6K.
Assuntos
PTEN Fosfo-Hidrolase/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/fisiologia , Treonina/metabolismo , Sequência de Bases , Biocatálise , Linhagem Celular Tumoral , Primers do DNA , Técnicas de Silenciamento de Genes , Humanos , Fosforilação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas c-akt/química , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Notch1/genéticaRESUMO
This article is a comprehensive clinical review of the initial evaluation of diabetic foot disease.