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Journal of Cellular Biochemistry

Flow cytometric detection of total and serine 473 phosphorylated Akt

Authors

  • Pier Luigi Tazzari,

    1. Servizio di Immunoematologia e Trasfusionale, Policlinico S.Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy
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  • Alessandra Cappellini,

    1. Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Sezione di Anatomia, Cell Signalling Laboratory, School of Pharmacy, Università di Bologna, via Irnerio 48, 40126 Bologna, Italy
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  • Roberta Bortul,

    1. Dipartimento di Morfologia Umana Normale, Università di Trieste, via Manzoni 16, 34138 Trieste, Italy
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  • Francesca Ricci,

    1. Servizio di Immunoematologia e Trasfusionale, Policlinico S.Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy
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  • Anna Maria Billi,

    1. Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Sezione di Anatomia, Cell Signalling Laboratory, School of Pharmacy, Università di Bologna, via Irnerio 48, 40126 Bologna, Italy
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  • Giovanna Tabellini,

    1. Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Sezione di Anatomia, Cell Signalling Laboratory, School of Pharmacy, Università di Bologna, via Irnerio 48, 40126 Bologna, Italy
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  • Roberto Conte,

    1. Servizio di Immunoematologia e Trasfusionale, Policlinico S.Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy
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  • Alberto M. Martelli

    Corresponding author
    1. Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Sezione di Anatomia, Cell Signalling Laboratory, School of Pharmacy, Università di Bologna, via Irnerio 48, 40126 Bologna, Italy
    2. Istituto di Citomorfologia Normale e Patologica del CNR, c/o IOR, via di Barbiano 1/10, 40137 Bologna, Italy
    • Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Sezione di Anatomia, Cell Signalling Laboratory, Università di Bologna, via Irnerio 48, 40126 Bologna, Italy.
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Abstract

The evaluation of regulatory proteins is important for biological studies and is also established as a prognostic marker for cancer diagnosis. Very recently, it has been highlighted that the serine/threonine kinase Akt plays a fundamental role in survival pathways and is also involved in the onset of resistance to anti-neoplastic drugs and ionizing radiation in cell lines derived from solid tumors. For its full activation Akt needs to be phosphorylated on Serine 473 residue. Molecules that are fundamental in determining resistance to therapeutic treatments might serve in the future as clinical markers to tailor therapy and/or predict treatment response. The aim of this study was to ascertain whether or not flow cytometric analysis of total Akt and of its form phosphorylated on Serine 473 could be related to standard techniques such as Western blotting with phosphospecific antibodies and in vitro kinase assay. To this end, we employed as experimental models HL-60 and PC-12 lines in which there is an enhancement of Akt activity. Our results showed that flow cytometry analysis, performed on fixed and permeabilized cells, correlated well with the results provided by in vitro activity assays and Western blots. Therefore, our findings might indicate that flow cytometric study of Akt (both total and phosphorylated) content may be applied in routine work for phenotyping of hematological and non-hematological neoplasias, and allow for its use as a useful marker for the classification and the prognosis of neoplastic diseases. J. Cell. Biochem. 86: 704–715, 2002. © 2002 Wiley-Liss, Inc.

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