Rapid detection of phosphotyrosine proteins by flow cytometric analysis in Bcr-Abl-positive cells.
BACKGROUND
Constitutive tyrosine phosphorylation derived from Bcr-Abl kinase activity is the major characteristic of Bcr-Abl positive cells. In this study, we developed a method to detect the phosphotyrosine proteins by flow cytometry and we asked whether phosphorylation was affected by imatinib mesylate treatment.


METHODS
Cells were treated or not with imatinib mesylate, fixed and permeabilized by PFA followed by saponin, then stained with anti-phosphotyrosine (p-tyr) monoclonal antibody and analyzed by flow cytometry.


RESULTS
Optimal staining parameters were performed with p-tyr antibody using K562 and LAMA84 lines that displayed high levels of tyrosine phosphorylation as compared to the control line, HL60. Tyrosine phosphorylation was inhibited by imatinib in a dose-dependent manner, but not modified by other inhibitors demonstrating that the staining detected is specific to Bcr-Abl phosphorylation. The staining of imatinib-resistant cell lines such as the mutated BaF/Bcr-AblT315I cell line or resistant CML patient cells, showed that hyperphosphorylation was not affected by imatinib treatment. In one CML patient, our technique permitted us to detect a small hyperphosphorylated population resistant to imatinib that appeared hyperphosphorylated and amplified at the time of relapse.


CONCLUSIONS
We have developed a flow cytometric technique presenting several advantages such as rapidity and sensitivity, which requires fewer cells than the Western blot.
Laboratoire Hématopoïèse Normale et Pathologique, FRE CNRS 2617, Université Victor Ségalen, rue Léo-Saignat, 33076 Bordeaux Cedex, France.
Vanessa
V
Desplat
Valérie
V
Lagarde
Francis
F
Belloc
Claudine
C
Chollet
Thibaut
T
Leguay
Jean-Max
JM
Pasquet
Vincent
V
Praloran
François-Xavier
FX
Mahon
D000818:Animals; D001549:Benzamides; D014408:Biomarkers, Tumor / Q000378:metabolism; D001854:Bone Marrow Cells / Q000187:drug effects / Q000473:pathology; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D016044:Fusion Proteins, bcr-abl / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D000068877:Imatinib Mesylate; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology / Q000627:therapeutic use*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000473:pathology; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology / Q000627:therapeutic use*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011743:Pyrimidines / Q000494:pharmacology / Q000627:therapeutic use*
D001549:Benzamides; D014408:Biomarkers, Tumor; D047908:Intracellular Signaling Peptides and Proteins; D010879:Piperazines; D011743:Pyrimidines; C015482:protein kinase modulator; D019000:Phosphotyrosine; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl