RESUMO
The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H(2)O(2)-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H(2)O(2) after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H(2)O(2) in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-ß inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H(2)O(2) production in AML cells is mediated by p22phox and is critical for STAT5 signalling.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Leucemia Mieloide Aguda/genética , NADPH Oxidases/metabolismo , Fator de Transcrição STAT5/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo , Benzoxazóis/farmacologia , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Imidazóis/farmacologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Pirróis/farmacologia , RNA Interferente Pequeno/genética , Fator de Transcrição STAT5/antagonistas & inibidores , Fator de Transcrição STAT5/genética , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Triazóis/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.
