Rapid detection of phosphotyrosine proteins by flow cytometric analysis in Bcr-Abl–positive cells
Article first published online: 5 OCT 2004
Copyright © 2004 Wiley-Liss, Inc.
Cytometry Part A
Volume 62A, Issue 1, pages 35–45, November 2004
How to Cite
Desplat, V., Lagarde, V., Belloc, F., Chollet, C., Leguay, T., Pasquet, J.-M., Praloran, V. and Mahon, F.-X. (2004), Rapid detection of phosphotyrosine proteins by flow cytometric analysis in Bcr-Abl–positive cells. Cytometry, 62A: 35–45. doi: 10.1002/cyto.a.20030
- Issue published online: 21 OCT 2004
- Article first published online: 5 OCT 2004
- Manuscript Accepted: 23 JAN 2004
- Manuscript Revised: 19 DEC 2003
- Manuscript Received: 23 MAY 2003
- Fondation de France
- Fondation pour la Recherche Medicale
- imatinib mesylate;
- tyrosine kinase;
- chronic myeloid leukemia
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.
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.
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.
We have developed a flow cytometric technique presenting several advantages such as rapidity and sensitivity, which requires fewer cells than the Western blot. © 2004 Wiley-Liss, Inc.