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

University of Bologna, Bolonia, Emilia-Romagna, 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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    • "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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    • "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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