[Show abstract][Hide abstract] ABSTRACT: 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-β 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jul 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Bcr-Abl causes chronic myelogenous leukemia, a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells. In this study, inducible expression of Bcr-Abl in TonB.210 cells is associated with increased production of intracellular reactive oxygen species (ROS), which is thought to play a role in survival signaling when generated at specific levels. Elevated ROS in Bcr-Abl-expressing cells were found to activate PI3k/Akt pathway members such as Akt and GSK3beta as well as downstream targets beta-catenin and Mcl-1. The activation of these proteins was inhibited by the flavoprotein inhibitor diphenyleneiodonium, which is commonly used to inhibit NADPH oxidase (Nox). This indicated that increased ROS might be related to increased activity of one member of the Nox family. Knock-down experiments using siRNA suggest that Nox-4 is the main source of increased ROS following Bcr-Abl expression. We showed that Bcr-Abl-induced ROS could also increase survival pathway signaling through redox inhibition of PP1alpha, a serine threonine phosphatase that negatively regulates the PI3k/Akt pathway. Overall our results demonstrate that Bcr-Abl expression increases Nox-4-generated ROS, which in turn increases survival signaling through PI3k/Akt pathway by inhibition of PP1alpha, thus contributing to the high level of resistance to apoptosis seen in these Bcr-Abl-expressing cells.
Full-text · Article · Apr 2009 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
[Show abstract][Hide abstract] ABSTRACT: There is increasing evidence within the literature that the decreased susceptibility of tumour cells to stimuli that induce apoptosis can be linked to their inherently increased redox potential. The review primarily focuses on the PI3-kinase/Akt pathway, and the multiple points along this signalling pathway that may be redox regulated. The PI3-kinase/Akt pathway can influence a cells' sensitivity to death inducing signals, through direct manipulation of apoptosis regulating molecules or by regulating the activity of key transcription factors. Proteins involved in the control of apoptosis that are directly regulated by the PI3-kinase/Akt pathway include caspase-9, Bad and the transcription factor GSK-3beta. Lately, it is becoming increasingly obvious that phosphatases are a major counter balance to the PI3-kinase/Akt pathway. Phosphatases such as PP2A and PP1alpha can dephosphorylate signalling molecules within the PI3-kinase/Akt pathway, blocking their activity. It is the balance between the kinase activity and the phosphatase activity that determines the presence and strength of the PI3-kinase/Akt signal. This is why any protein modifications that hinder dephosphorylation can increase the tumours survival advantage. One such modification is the oxidation of the sulphydryl group in key cysteine residues present within the active site of the phosphatases. This highlights the link between the increased redox stress in tumours with the PI3-kinase/Akt pathway. This review will discuss the various sources of reactive oxygen species within a tumour and the effect of these radicals on the PI3-kinase/Akt pathway.