Detection of serine 473 phosphorylated Akt in acute myeloid leukaemia blasts by flow cytometry

Servizio di Immunoematologia e Trasfusionale, Policlinico S. Orsola-Malpighi, via Irnerio 48, 40126 Bologna, Italy.
British Journal of Haematology (Impact Factor: 4.71). 10/2004; 126(5):675-81. DOI: 10.1111/j.1365-2141.2004.05121.x
Source: PubMed


The phosphoinositide 3-kinase/Akt signalling pathway is a recently recognized important parameter in the prognosis and the response to treatment of acute myeloid leukaemia (AML). Akt kinase is activated by phosphorylation on Thr 308 and Ser 473. Active Akt promotes cell growth and survival to apoptotic insults. Thus, it seems important to evaluate Akt phosphorylation in AML blasts. This work aimed to establish whether it was possible to detect Akt phosphorylation on Ser 473 of AML blasts by means of flow cytometry. High levels of Akt activity and phosphorylation were detected in 13 of 15 cases of AML. Flow cytometric analysis revealed similar patterns of Ser 473 expression as was observed with Akt kinase activity and Western blot analysis of Thr 308 and Ser 473 phosphorylation. Double immunostaining enabled the simultaneous flow cytometric detection of an AML-associated antigen (CD33) and Ser 473 phosphorylated Akt in leukaemic blast populations. Our results indicate that flow cytometry enabled the rapid and quantitative assessment of Ser 473 phosphorylated Akt of AML blasts that, when used in combination with cell surface staining, can provide more accurate phenotyping of AML blasts.

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Available from: Irina Mantovani, Aug 25, 2015
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    • "The phosphorylation status of Akt in three different types of leukemia presents an interesting case. Tazzari et al. (2004) reported high levels of S473 phosphorylation in acute myeloid leukemia blasts (AML blasts), similarly Nyakern et al. (2006) reported high levels of Akt S473 phosphorylation in mononuclear cells from bone marrow of the patients with high-risk myelodysplastic syndrome (MDS) when compared to normal or low risk MDS patients; Gallay et al. (2009) on the other hand, reported higher T308 phosphorylation in patients with AML, which was shown to be associated with high-risk cytogenetics and poor overall survival. Although apparently contradictory, the results reflect the status of proliferation of the cells examined; actively proliferating AML cases have high T308, while the AML blasts and MDS, which are poorly dividing cells have high levels of S473. "
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    ABSTRACT: The serine threonine protein kinase, Akt, is at the central hub of signaling pathways that regulates cell growth, differentiation, and survival. The reciprocal relation that exists between the two activating phosphorylation sites of Akt, T308 and S473, and the two mTOR complexes, C1 and C2, forms the central controlling hub that regulates these cellular functions. In our previous review "PI3Kinase (PI3K)-AKT-mTOR and Wnt signaling pathways in cell cycle" we discussed the reciprocal relation between mTORC1 and C2 complexes in regulating cell metabolism and cell cycle progression in cancer cells. We present in this article, a hypothesis that activation of Akt-T308 phosphorylation in the presence of high ATP:AMP ratio promotes the stability of its phosphorylations and activates mTORC1 and the energy consuming biosynthetic processes. Depletion of energy leads to inactivation of mTORC1, activation of AMPK, FoxO, and promotes constitution of mTORC2 that leads to phosphorylation of Akt S473. Akt can also be activated independent of PI3K; this appears to have an advantage under situations like dietary restrictions, where insulin/insulin growth factor signaling could be a casualty.
    Frontiers in Oncology 06/2013; 3:165. DOI:10.3389/fonc.2013.00165
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    • "Various studies have demonstrated that many components of the Akt signaling pathway are constitutively active in a wide range of human tumors, especially in leukemia45. Leukemia cells display increased expression of a phosphorylated form of Akt compared with granulocytes46. Additionally, the phosphorylation levels of GSK3β and S6k1, which stimulate protein synthesis and cell survival as downstream effectors of Akt, are also high in leukemia cells45. "
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    ABSTRACT: Gambogic acid (GA) is the main active ingredient of gamboge, a brownish to orange dry resin secreted from Garcinia hanburyi, a plant that is widely distributed in nature. Recent in vitro and in vivo studies have demonstrated that GA exerts potent antitumor effects against solid tumors of various derivations, and its antitumor mechanisms have been thoroughly investigated. On the other hand, normal cells remain relatively resistant to GA, indicating a therapeutic window. GA is currently in clinical trials in China. Over the last decade, our laboratory demonstrates that GA exhibits potent anticancer activities against hematological malignancies. This review focuses on the new mechanisms through which GA inhibits proliferation and induces apoptosis in malignant hematological cells. These include the regulation of expression and intracellular positioning of nucleoporin and nucleophosmin; downregulation of steroid receptor coactivator-3 (SRC-3) and its downstream proteins; upregulation of death inducer-obliterator (DIO-1); downregulation of HERG potassium channel; as well as induction of reactive oxygen species (ROS) accumulation.
    Acta Pharmacologica Sinica 12/2012; 34(2). DOI:10.1038/aps.2012.163 · 2.91 Impact Factor
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    • "Quantitative flow cytometry appears particularly well suited for this kind of analysis, because it offers obvious advantages over other techniques (western blot, for example), including quickness, a much lower number of cells required to perform the assay, and the possibility of identifying different subclones in the leukemic population by co-immunostaining with multiple antibodies to surface antigens. Accordingly, flow cytometry is rapidly becoming the choice analytical technique to study PI3K/Akt/mTOR pathway activation in AML patients [70, 133, 152, 153]. Another promising quantitative technique requiring a limited number of cells, which has been already applied to the study of AML patients samples, is represented by reverse-phase protein arrays [74]. "
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    ABSTRACT: The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth, and survival under physiological conditions. However, aberrant PI3K/Akt/mTOR signaling has been implicated in many human cancers, including acute myelogenous leukemia (AML). Therefore, the PI3K/Akt/mTOR network is considered as a validated target for innovative cancer therapy. The limit of acceptable toxicity for standard polychemotherapy has been reached in AML. Novel therapeutic strategies are therefore needed. This review highlights how the PI3K/Akt/mTOR signaling axis is constitutively active in AML patients, where it affects survival, proliferation, and drug-resistance of leukemic cells including leukemic stem cells. Effective targeting of this pathway with small molecule kinase inhibitors, employed alone or in combination with other drugs, could result in the suppression of leukemic cell growth. Furthermore, targeting the PI3K/Akt/mTOR signaling network with small pharmacological inhibitors, employed either alone or in combinations with other drugs, may result in less toxic and more efficacious treatment of AML patients. Efforts to exploit pharmacological inhibitors of the PI3K/Akt/mTOR cascade which show efficacy and safety in the clinical setting are now underway.
    Oncotarget 06/2010; 1(2):89-103. DOI:10.18632/oncotarget.114 · 6.36 Impact Factor
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