Flow cytometric detection of total and serine 473 phosphorylated Akt

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.