The Philadelphia (Ph) chromosome caused by a reciprocal translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)] is the hallmark of chronic myeloid leukemia (CML). The translocation leads to the expression of a chimeric protein Bcr-Abl with deregulated tyrosine kinase activity (1), which is responsible for leukemogenesis in vitro and in vivo (2). This marker can be found in almost all cases (95%) of CML and in 25% of adult and 5% of children with acute lymphoblastic leukemia (ALL) (3). CML progresses through three clinical stages: a chronic phase (CP), an accelerated phase (AP), and finally a fatal blast crisis (BC). The BCR-ABL oncogene is expressed at all stages, but BC is characterized by multiple additional genetic and molecular changes. The transforming effect of Bcr-Abl mainly induces abnormal proliferation, resistance to apoptosis and modifications of cell adhesion, and genetic instability. Indeed, Bcr-Abl phosphorylates many cellular targets, leading to the activation of intracellular signaling pathways such as Ras (4), Jak/STAT (5), and Akt/PI-3 kinase (6) pathways.
Imatinib mesylate (Gleevec or Glivec, formerly STI571) is a Bcr-Abl tyrosine kinase inhibitor that specifically targets the ATP-binding site to the Abl kinase domain at micromolar concentrations, thereby selectively inducing inhibition of the growth of Bcr-Abl–positive cells (7, 8) and apoptosis in leukemic cells (9–11). The remarkable effects of imatinib in CML have been clinically demonstrated in CP and confirmed in large trials (12). However, drug resistance is now a critical problem, particularly in the advanced phase of the disease. Patient remissions with advanced CML or ALL are transient, and most patients relapse despite continued treatment. To investigate resistance to this inhibitor, several groups have reported the isolation of imatinib-resistant Bcr-Abl–positive cells (13–16). Several mechanisms of resistance have been proposed including Bcr-Abl overexpression or increased expression of the mdr-1 gene encoded Pgp (13, 17). Recently, analysis of leukemic cells from patients with CML showed that acquired resistance to imatinib was due either to a point mutation in the kinase domain of Bcr-Abl or amplification of the BCR-ABL gene (18–21).
One of main characteristics of Bcr-Abl is the constitutive ability to hyperphosphorylate the cellular tyrosines of several protein substrates. Tyrosine phosphorylation of proteins has previously been investigated by Western blot in leukemic cell lines (22, 23), but this technique requires large quantities of cells that are difficult to obtain from patient samples. Protein tyrosine phosphorylation was previously studied by flow cytometry to study activation of T lymphocytes (24). We have now developed a method to detect the phosphotyrosine proteins by flow cytometry in order to analyze the level of the tyrosine hyperphosphorylation of cellular proteins induced by Bcr-Abl. In the present work, we describe how this technique may be used to distinguish Bcr-Abl–positive cells from the others and to detect imatinib-resistant cells in patients.
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The phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth. Its dysregulation can result in various types of cancer. Bcr-Abl, which is the hallmark of CML, constitutively induces the phosphorylation of a large panel of proteins, activating the multiple signaling pathways responsible for protection against apoptosis, stimulation of growth factor-independent proliferation, and alterations of cellular adhesion. Using Western blot analysis, CML cells exhibit a very high level of phosphorylation compared to Bcr-Abl negative cells (28). We utilized these properties to set up a swift flow cytometric procedure for differentiating positive and negative cells.
For the detection of cellular phosphotyrosine proteins, two different anti-phosphotyrosine monoclonal antibodies were used: clone PY99 for cytometric analysis, and clone 4G10 for Western blot analysis. Indeed, flow cytometry staining performed with 4G10 showed the detection of tyrosine phosphorylation, but with less affinity than PY99. In contrast, detection of phosphotyrosine using the PY99 antibody for Western blot analysis was not better than when 4G10 was used.
So, we performed an intracytoplasmic staining with clone PY99, using Bcr-Abl–positive and –negative cell lines. We found that Bcr-Abl–positive cell lines were hyperphosphorylated as compared to Bcr-Abl–negative cell lines. Treatment of cells with imatinib induced a decrease of tyrosine phosphorylation levels. The same quantitative results were obtained with the Western blot technique using the clone 4G10. An inhibition in tyrosine phosphorylation by imatinib was also detected in Bcr-Abl–positive cell lines. In contrast to flow cytometry, this technique permitted the detection of several bands corresponding to different substrate proteins; the band corresponding to the p210 Bcr-Abl protein was dephosphorylated by imatinib in a dose-dependent manner, and phosphorylation of STAT5 was shown by a specific Western blot analysis.
To prove the specificity of our detection method, Bcr-Abl–positive cell lines were incubated with alkaline phosphatase and pV, respectively inducing a dephosphorylation and an increase in phosphorylation of cells, demonstrating that our staining is able to detect the fluctuation in phosphorylation/dephosphorylation equilibrium in cells. Then we used more specific inhibitors, such as PKC412 (a PKC inhibitor) and SU5614 (a tyrosine kinase inhibitor of VEGF receptor), which revealed no modification in phosphorylation level of cells. These results were expected, since it has been reported that SU5614 (0.1–1.0 μM) did not show an inhibitory effect on the BaF/Bcr-Abl cell line (30).
Taken together, these results showed that our flow cytometric assay method appears suitable for measuring cellular phosphotyrosine levels and for studying imatinib resistance.
Resistant cell lines, generated to study the mechanisms of resistance developed by leukemic cells, displayed an elevated phosphotyrosine content, even with high doses of imatinib and independent of the mechanism of resistance (Bcr-Abl hyperexpression or mutation in the tyrosine kinase domain). There is a relation between tyrosine phosphorylation and imatinib resistance. As shown in Figure 6, we observed in vitro, using phosphorylation study by flow cytometry, that mutation T315I is very resistant to imatinib. These results confirm that an inhibitory concentration of imatinib could not be reached in the context of clinically achievable values.
Study of tyrosine phosphorylation of CML patients' mononuclear cells sensitive or resistant to imatinib demonstrated that: 1) the cells are hyperphosphorylated as Bcr-Abl–positive cell lines, and 2) the treatment of cells with imatinib induced an inhibition of tyrosine phosphorylation in sensitive patient cells, but did not induce a decrease in the level of tyrosine phosphorylation for resistant patients, demonstrating the resistance of these cells for the treatment. Here, the mechanism of resistance remains to be determined, since it results neither from mutations in ATP binding, nor from Bcr-Abl amplification or Pgp overexpression (data not shown).
In one of the CML patients studied, a resistant hyperphosphorylated population was detected before imatinib treatment. At the time of the relapse, this patient exhibited a single resistant hyperphosphorylated population, a finding that could predict the patient resistance to the treatment, which is interesting from a clinical point of view.
In ALL Ph+ patients, the leukocyte population was distinguished from the blastic population and their quantification was allowed. Addition of imatinib had no effect on the level of tyrosine phosphorylation in these two populations, thereby confirming the resistance of the Bcr-Abl–positive population and the selectivity of the inhibitor.
In conclusion, we describe here a rapid and convenient method to measure global cellular phosphotyrosine levels by flow cytometry. This technique offers several advantages compared to Western blot analysis. Its rapidity and sensitivity make it particularly convenient for the routine use and processing of a large number of samples. It also differentiates cell populations, and can be used with heterogeneous samples in combination with other fluorescent markers. On the other hand, the technique obviously cannot identify the proteins phosphorylated by Bcr-Abl, unlike Western blot, nor can it allow the identification of numerous sites of tyrosine phosphorylation, unlike mass spectrometry (33, 34). The technique could also be used to detect other activated tyrosine kinases involved in other malignancies, as has been reported for c-kit (35) and Flt3 in AML (36), and for Braf in melanoma (37).