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Flow cytometric detection of total and serine 473 phosphorylated Akt
Pier Luigi Tazzari1, Alessandra Cappellini, Roberta Bortul
1Servizio di Immunoematologia e Trasfusionale, Policlinico S.Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy.
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.
Insights
Flow cytometry can effectively measure total and phosphorylated Akt protein levels, crucial for cancer prognosis. This method correlates well with traditional techniques, offering a potential tool for cancer phenotyping and treatment response prediction.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Regulatory proteins like Akt are vital in biological studies and cancer prognosis.
- The serine/threonine kinase Akt is implicated in cell survival, drug resistance, and radiation resistance in solid tumors.
- Full Akt activation requires phosphorylation at the Serine 473 residue.
Purpose of the Study:
- To evaluate the correlation between flow cytometric analysis of total Akt and phosphorylated Akt (p-Akt) and standard methods.
- To assess the utility of flow cytometry for Akt evaluation in cancer research and diagnostics.
Main Methods:
- Utilized HL-60 and PC-12 cell lines with enhanced Akt activity as experimental models.
- Employed flow cytometry on fixed and permeabilized cells to measure total and phosphorylated Akt.
- Compared flow cytometry results with Western blotting using phosphospecific antibodies and in vitro kinase assays.
Main Results:
- Flow cytometry analysis of total and phosphorylated Akt demonstrated strong correlation with Western blot and in vitro kinase assay results.
- The findings support the reliability of flow cytometry for assessing Akt status in cell lines.
Conclusions:
- Flow cytometry is a viable method for evaluating total and phosphorylated Akt.
- This technique may be applicable in routine phenotyping of hematological and non-hematological neoplasias.
- Akt flow cytometry could serve as a valuable marker for cancer classification and prognosis.