Single cell analysis of phosphoinositide 3-kinase/Akt and ERK activation in acute myeloid leukemia by flow cytometry.
OBJECTIVE
Abnormal activation of several signal transduction pathways such as phosphoinositide 3-kinase (PI3K) and MAP kinases has been reported in acute myeloid leukemia (AML). To test new targeted therapeutics, it is critical to develop sensitive analytical tools to detect abnormal activation of these pathways and to monitor their inhibition in response to treatment.


METHODS
We analyzed Akt and ERK phosphorylation in 32 samples from patients using western blot and a two-color flow cytometry protocol using CD34. To circumvent the CD34 negative AML found in our series, we developed a two-color protocol using CD45 to isolate the blast cell population. Finally, a four-color protocol was used to detect phosphorylation in an enriched population of AML stem cells.


RESULTS
We compared western blot analysis and flow cytometry for the detection of PI3K/Akt and ERK activation and found a 100% correlation between the two techniques in a series of 32 AML samples. Using a flow cytometry protocol, we were able to analyze all the patients' samples, even those with low blast infiltration or CD34 negative blast cells. We were also able to detect the phosphorylated proteins in the most immature blast cell population with the CD34+ CD38-/low CD123+ phenotype. Interpretations and


CONCLUSIONS
Our study shows that flow cytometry is a reliable method for detecting Akt and ERK phosphorylation in all patients' samples. Activation can also be detected in the most immature blast cells, which represent exquisite target cells for new therapeutics.
Bardet
Valérie
V
Université Paris-Descartes, Faculté de Médecine, Institut Cochin (INSERM U567, CNRS-UMR 8104), Paris, France. valerie.bardet@cch.aphp.fr
Tamburini
Jerome
J
Ifrah
Norbert
N
Dreyfus
François
F
Mayeux
Patrick
P
Bouscary
Didier
D
Lacombe
Catherine
C
D000208:Acute Disease; D015703:Antigens, CD / Q000032:analysis; D001752:Blast Crisis; D001854:Bone Marrow Cells / Q000201:enzymology / Q000473:pathology; D004789:Enzyme Activation; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007951:Leukemia, Myeloid / Q000097:blood / Q000201:enzymology* / Q000276:immunology / Q000473:pathology; D020930:MAP Kinase Kinase Kinases / Q000097:blood*; D009000:Monocytes / Q000201:enzymology; D019869:Phosphatidylinositol 3-Kinases / Q000097:blood*; D051057:Proto-Oncogene Proteins c-akt / Q000097:blood*
D015703:Antigens, CD; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D020930:MAP Kinase Kinase Kinases