Inhibition of tyrosine phosphorylation in primary cells by imatinib mesylate. (A) Median levels of tyrosine phosphorylation in cytogenetic responders (□) and nonresponders (▪). 104 to 105 CD34+ cells per data point were treated for 2 hours with a range of imatinib mesylate concentrations, fixed in 1% paraformaldehyde, permeabilized with 0.3% saponin, and incubated with an antiphosphotyrosine (PY99; Santa Cruz, Holly Ditch Farm, United Kingdom) and a secondary fluorescent antibody (goat F(ab′)2 antimouse; Caltag, Silverstone, United Kingdom). The dose of imatinib mesylate which best distinguishes the 2 groups was found to be 20 μM. (B) Median percentage tyrosine phosphorylation of imatinib mesylate-treated cells from cytogenetic responders (R) and nonresponders (NR) compared with cells not exposed to the drug. The data show a significant difference between the median tyrosine phosphorylation of the 2 groups (Mann-Whitney test, P = .034). In about half of the patients responding to imatinib mesylate the tyrosine phosphorylation levels upon in vitro exposure to the inhibitor fell below 55% (dotted line) of that in the nonexposed cells, whereas this level of reduction was not achieved in any of the nonresponders. (C) Illustrative flow cytometric profile of 1 responder (top) and 1 nonresponder (bottom). Cells stained with the isotype control antibody (gray line) are used to adjust the flow cytometer settings, whereas cells not exposed to imatinib mesylate serve as a phosphotyrosine staining baseline for all measurements (black line). Imatinib mesylate-treated cells (shaded curve) from patients who eventually responded to imatinib mesylate show a clearly visible shift to the left, while the shift is significantly smaller for the nonresponders (as shown by the distance between the arrows pointing to the peak fluorescence of the exposed and nonexposed cells). (D) Comparison of CD34 cells used fresh or after 1 round of freezing for the assessment of total phosphotyrosine phosphorylation. From 7 extra patients, CD34+ cells from freshly collected blood were divided into 2 aliquots: 1 was placed in culture for the assay on “fresh” cells, and the second was cryopreserved and thawed later on for the same test. Each line depicts 1 paired sample of cells.
