Rapid detection of phosphotyrosine proteins by flow cytometric analysis in Bcr-Abl-positive cells.

Cytometry A 2004 Nov;62(1):35-45

Laboratoire Hématopoïèse Normale et Pathologique, FRE CNRS 2617, Université Victor Ségalen, rue Léo-Saignat, 33076 Bordeaux Cedex, France.

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.

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http://dx.doi.org/10.1002/cyto.a.20030DOI Listing
November 2004
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References

(Supplied by CrossRef)

Lugo et al.
Science 1990

Daley et al.
Science 1990

Ghaffari et al.
Leukemia 1999

Cortez et al.
Oncogene 1996

Ilaria et al.
J Biol Chem 1996

Skorski et al.
EMBO J 1997

Druker et al.
Nat Med 1996

Deininger et al.
Blood 1997

Gambacorti-Passerini et al.
Blood Cells Mol Dis 1997

Dan et al.
Cell Death Differ 1998

Fang et al.
Blood 2000

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