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Keywords:

  • hyperphosphorylation;
  • imatinib mesylate;
  • Bcr-Abl;
  • tyrosine kinase;
  • chronic myeloid leukemia

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

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. © 2004 Wiley-Liss, Inc.

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Cell Lines

The human cell lines K562, LAMA84, LAMA84-r, and HL60 were grown in RPMI 1640 medium (Life Technology, Cergy-Pontoise, France) supplemented with 10% fetal calf serum (FCS), antibiotics (penicillin, streptomycin), and L-glutamine (complete medium referred to as RF-10) (13). A murine BaF/3 cell line was transfected by electroporation with wild-type Bcr-Abl pEYK expression vector and T315I mutated Bcr-Abl pEYK vector (plasmid kindly provided by George Daley, Whitehead Institute, Cambridge, UK), named respectively, BaF/Bcr-Abl-wt and BaF/Bcr-Abl-T315I (Leguay et al., unpublished results). These lines were then cultured in RF-10.

Patient Samples

Peripheral blood and/or bone marrow cells from CML patients treated with imatinib (600–800 mg) in the Novartis protocols for accelerated blast and phase crisis (102/115 and 109/114 (www.glivec.com)) were obtained before treatment and at the time of relapse. None of these patients exhibited amplification of the BCR-ABL gene (detected by FISH analysis) or point mutations in their tyrosine kinase domain that could account for the imatinib-resistant phenotype studied by sequencing (19, 25). For this study, total RNA was extracted from patients' mononuclear cells then reverse transcribed. PCR was performed to amplify a 1,298-bp fragment including the entire tyrosine kinase domain. Sequencing of amplified cDNA was performed using the ABI Prism dRhodamine Terminator Ready Reaction kit on the ABI Prism 377 (Perkin Elmer, Montigny-Le-Bretonneux, France). Sequence analysis was performed in reference to the ABL type 1a gene sequence (GenBank accession number M14752). Peripheral blood mononuclear cells were isolated by separation on a Ficoll gradient (Eurobio, les Ulis, France) (1,500 rpm, 20 min, room temperature), then frozen.

Reagents

Alkaline phosphatase and saponin were purchased from Sigma (Saint Quentin Fallavier, France). Imatinib mesylate was a gift from Novartis (Basel, Switzerland) and was solubilized in sterilized distilled water to make a 1 μM solution. PKC 412 and SU5614 were purchased from Novartis and Calbiochem (Fontenay sous Bois, France), respectively.

Vanadate peroxide (pV) was prepared by reacting 10 mM vanadate solution with 3 mM H2O2 for 15 min at 37°C. The concentration of vanadate peroxide is denoted by the vanadate concentration added to the mixture.

Antibodies

Phosphotyrosine (p-tyr) mouse monoclonal antibody clone PY99 was purchased from Santa Cruz Biotechnology (Heidelberg, Germany), p-tyr mouse monoclonal antibody clone 4G10 and mouse IgG2b antibody were provided from Pharmingen (BD Biosciences, Le-Pont-de-Claix, France). Fluorescein isothiocyanate-conjugated (FITC) goat anti-mouse (GAM) monoclonal antibody was from Becton Dickinson (Le-Pont-de-Claix, France). Anti-STAT5 monoclonal antibody was obtained from Santa Cruz Biotechnology, anti-phospho-STAT5 polyclonal antibody was obtained from Cell Signaling Technology (Ozyme, Saint Quentin en Yvelines, France), and anti-actin polyclonal antibody was from Sigma (France).

Phosphotyrosine Staining

Cell lines or mononuclear patient cells (5 × 105) were incubated for 2 h (37°C, 5% CO2) with increasing doses of imatinib mesylate (0, 0.1, 0.5, 1, 5, 10, 50, and 100 μM) in RF-10 (1 ml). Then, they were washed twice in ice-cold phosphate-buffered saline (PBS) and fixed in 1% paraformaldehyde (PFA)/PBS for 10 min at 4°C. After washing in PBS, cells were permeabilized with saponin (0.3% in PBS) and incubated with PY99 mouse monoclonal antibody (200 ng/ml in a constant reaction volume of 100 μl) for 1 h at 4°C as well as with the relevant isotypic control IgG2b. Then, cells were washed (PBS/0.3% saponin) and were stained by FITC-GAM monoclonal antibody as secondary reagent for 45 min at 4°C in the dark. After washing, cells were recovered in 500 μl of PBS/0.3% saponin and submitted to flow cytometric analysis.

Flow Cytometry

Flow cytometry analysis was performed with a Beckman Coulter Epics XL flow cytometer (Beckman-Coulter, Villepinte, France) with 440/25, 525/20, and 675 nm/25 nm band-pass (BP) filters for emission capture in blue, green and red ranges. Experiments were analyzed using the Expo 32 software (Beckman Coulter, Villepinte, France). For each experiment, cell volume and light scatter were used to gate out debris and cell death.

Western Blot

Protein lysates were prepared according to the method of Kabarowski et al. (26) with minor modifications. Protein concentrations were determined by the Bradford method (BIO-RAD, Marnes-La-Coquette, France). Approximately 50–100 μg proteins were resolved on 12% or 6% polyacrylamide gel. After electrophoresis, proteins were transferred to a PVDF membrane by a semidry electrophoretic transfer. The blots were blocked in 5% milk/TBS 1× (except for anti-phospho-STAT5 antibody when blots were blocked in 5% BSA/TBS 1×-tween) at room temperature with gentle shaking. They were then incubated with the respective antibody (1:1,000 dilution) followed by an anti-rabbit or anti-mouse peroxidase-conjugated secondary IgG antibody. The reaction was revealed with an enhanced chemoluminescence detection (ECL) method (Western Lightning Chemiluminescence Reagent Plus; Perkin Elmer, Montigny-Le-Bretonneux, France).

Images were developed on Hyperfilm (Amersham Biosciences, Orsay, France).

Data Analysis

The p-tyr content was defined as the mean fluorescence intensity (MFI) of the p-tyr–labeled sample minus the MFI of the corresponding isotypic control. The relative p-tyr content of imatinib-treated samples were normalized as follows: p-tyr content of treated sample × 100/p-tyr content of untreated sample. IC50 (corresponding to a 50% reduction in p-tyr content) was deduced from a curve showing the relative content as a function of imatinib concentrations. Statistical analysis was performed with Microsoft Excel 2000 (Microsoft, Redmond, WA).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Optimal Staining Antibody Concentration

To determine the appropriate working dilution of anti-p-tyr monoclonal antibody (α-PY99), cells were stained with increasing concentrations of α-PY99 and a constant amount of secondary antibody as described in Materials and Methods. The α-PY99 concentration was varied from 50 to 2000 ng/ml in a constant reaction volume of 100 μl. As shown in Figure 1, saturation binding of FITC-α-PY99 appears to be optimal at 200 ng/ml with no significant change up to the maximal concentration tested. Therefore, all experiments were performed with 200 ng/ml of α-PY99. Experiments were also performed using PE-conjugated antibody without improvement in results (data not shown).

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Figure 1. Optimal fixation of antiphosphotyrosine monoclonal antibody (PY99) binding. K562 cells were fixed in PFA, permeabilized in saponin, then stained with PY99 antibody. The fluorescence intensity was calculated as described in Materials and Methods.

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We stained Bcr-Abl–positive cell lines LAMA84 and K562 and a Bcr-Abl–negative cell line HL60, used as a control. This line exhibits only a slight tyrosine phosphorylation level compared to the isotypic control (Fig. 2). By contrast, LAMA84 and K562 show an overphosphorylation of tyrosine compared to HL60 cells (Fig. 2). MFIs were 2.72 ± 0.48 and 2.5 ± 0.32 for LAMA84 and K562, respectively, while HL60 was closed to the isotypic control (MFI of 0.7 ± 0.2 arbirary units).

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Figure 2. Detection of phosphotyrosine in leukemic cell lines. Bcr-Abl–negative cell line (HL60) and Bcr-Abl–positive cell lines (K562, LAMA84) were stained with p-tyr antibody (PY99) and analyzed by flow cytometry. One representative experiment out of four is shown. A negative isotypic control (IgG) was also performed.

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Effect of Imatinib on Tyrosine Phosphorylation

Because imatinib mesylate specifically reduces the tyrosine kinase activity of Bcr-Abl, we investigated its effect on the level of tyrosine phosphorylation in Bcr-Abl–positive cell lines. After incubation with increasing doses of imatinib mesylate (0, 0.1, 0.5, 1, 5, 10, 50, and 100 μM), the fluorescence intensity of the p-tyr staining decreased in a dose-dependent manner until there was a 79% reduction in green fluorescence intensity at the highest concentration (Fig. 3A). IC50 corresponded to 8.2 with a standard deviation of 2.1. Treatment of cells with higher doses of imatinib (200, 300, 400, and 500 μM) induced cell death by toxicity, which prevent the staining (data not shown). To confirm our results, we performed a Western blot analysis using a p-tyr monoclonal antibody, 4G10. Before preparation of protein lysates, cells were incubated with imatinib for 2 h in the same conditions as for cytometric experiments. Addition of imatinib induced an inhibition of tyrosine phosphorylation in a dose-dependent manner (Fig. 3B). Recently, it was reported (27) that phosphorylation of STAT5 is an appropriate measure of Bcr-Abl kinase activity. To complement our analysis, we performed Western blot analysis, detecting STAT5 phosphorylated (p-STAT5) and STAT5. As expected, imatinib treatment induced an inhibition in phosphorylation of STAT5, while STAT5 expression was equal to the imatinib concentrations. The results confirm those found with Western blot by other groups, thereby validating the method (13, 28).

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Figure 3. Inhibition of tyrosine phosphorylation in LAMA84 cell line by imatinib. A: Cells were incubated for 2 h with increasing doses of imatinib (0–100 μM), then stained with p-tyr antibody as described in Materials and Methods, and then analyzed by flow cytometry. B: Western blot analysis: 50 to 100 μg of total cellular proteins were harvested from cells grown with different concentrations (0–10 μM) of imatinib for 2 h. The Western blot membranes were probed with the antibodies indicated. Actin was used as control. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com].

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Specificity of the Staining

The phosphate level in tyrosine residues of a protein results from a balance between a phosphorylation reaction catalyzed by tyrosine kinases and a dephosphorylation reaction mediated by tyrosine phosphatases. We tested whether specific inhibitors of these enzymes could modify this equilibrium. After fixation and permeabilization as described in Materials and Methods, K562 cells were incubated with alkaline phosphatase (20 units) for 1 and 2 h. Then cells were analyzed for phosphotyrosine staining. The results of one experiment are presented in Figure 4. Incubation of cells with phosphatase induced a time-dependent decrease in tyrosine phosphorylation levels. A total of 50% of cells were dephosphorylated after 1 h of treatment and almost a totality of cells were dephosphorylated after 2 h (Fig. 4A).

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Figure 4. FACS analysis of the effects of tyrosine kinase and tyrosine phosphatase inhibitors on phosphotyrosine levels in K562 cells. A: Cells were fixed with PFA 1%, permeabilized with 0.3% saponin, and incubated with alkaline phosphatase (20 units) for 1 and 2 h at 37°C, then stained with p-tyr antibody. B: Before phosphotyrosine staining, cells were incubated with pV (100 μM) in culture medium for 15 min at 37°C and then processed for flow cytometric analysis.

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In contrast, the treatment of cells with a tyrosine phosphatase inhibitor, pV (100 μM), for 15 min induced a phosphorylation as shown by an increase of green fluorescence intensity (2.3 versus 3.6 arbirary units) after phosphotyrosine staining (Fig. 4B).

The effects of specific inhibitors were also investigated on LAMA84 and K562 cell lines. Cells were incubated with increasing doses of other inhibitors such as PKC412 (0.5–100 μM), which is an inhibitor of PKC (a serine/threonine-directed protein kinase) (29), or SU5614 (0.5–10 μM), which is a potent inhibitor of tyrosine kinase VEGF and PDGF receptors (30). No effect on phosphotyrosine levels in either of these cell lines was detected (Fig. 5). However, a small inhibition was observed with PKC412 (between 10 to 100 μM), this is probably due to a nonspecific effect or to the inhibition of PKC (29).

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Figure 5. Analysis of effects of two specific tyrosine kinase inhibitors on phosphotyrosine levels in K562 cell line. Cells were incubated for 2 h with increasing doses of PKC412 (0–100 μM) and SU5614 (0–10 μM), then stained with p-tyr antibody as described in Materials and Methods, and analyzed by flow cytometry.

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Tyrosine Hyperphosphorylation Was Not Affected by Imatinib in Resistant Cell Lines

Resistant leukemic cells were previously generated (13–15) to study the mechanisms of resistance developed by leukemic cells. We studied the effect of imatinib on overexpressing Bcr-Abl cell lines (LAMA84-r and AR230-r). The doses of the inhibitor necessary to inhibit overall tyrosine phosphorylation were higher for the resistant cells than for the respective parental clone (data not shown).

Some imatinib-resistant patients exhibited an other mechanism of resistance, i.e., the presence of mutations in the ATP binding site of Abl. There is evidence for a point mutation resulting in a Thr[RIGHTWARDS ARROW]Ile change at Abl 315 in the kinase domain of the Bcr-Abl protein, which has previously been shown to be important for imatinib binding (18). We investigated the role of this mutation on the sensitivity to the inhibition of tyrosine phosphorylation by using the murine BaF/3 cell line transfected with Bcr-Abl-T315I in comparison to BaF/Bcr-Abl-wt transfected with wt-Bcr-Abl (imatinib-sensitive). As shown in Figure 6, imatinib decreased the level of tyrosine phosphorylation in the sensitive line (IC50 = 8 μM), while in the mutated line, the decrease was lower (IC50 = 21 μM ± 2.85). This result is consistent with the imatinib resistance of the BaF/Bcr-Abl-T315I cell line, and the detection of this kind of resistance by flow cytometry.

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Figure 6. Detection of resistance to imatinib by analysis of phosphotyrosine in BaF/Bcr-Abl cell lines. After 2 h of incubation with increasing doses of imatinib (0–100 μM), BaF/Bcr-Abl-wt cells (A) and BaF/Bcr-Abl-T315I cells (B) were labeled with p-tyr antibody (PY99) and analyzed by flow cytometry. Both cell lines contain multiple copies of Bcr-Abl because they were obtained after transfection (13). One experiment representative of four is shown. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com].

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Tyrosine Phosphorylation Study in CML Patient Cells

Since this flow cytometric method successfully estimates the tyrosine phosphorylation level induced by Bcr-Abl, we used it to study the level of tyrosine phosphorylation in cells from BC phase patients, since resistance appears at this moment (31). Analysis of mononuclear patient cells showed different patterns of phosphorylation, but all the samples analyzed exhibited 90% of the blast cells. Figure 7A illustrates the response of CML patient cells sensitive to imatinib and incubated with increasing doses of imatinib (0–50 μM). The cells were hyperphosphorylated and imatinib decreased tyrosine phosphorylation in a dose-dependent manner. By contrast, some patient cells were refractory and remained hyperphosphorylated even with high doses of the inhibitor, thus suggesting their resistance (Fig. 7B).

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Figure 7. Analysis of phosphotyrosine expression in CML patient cells. Mononuclear cells from CML patients in BC were labeled with p-tyr antibody (PY99) after 2 h of treatment with increasing doses of imatinib (0–100 μM). A: Sensitive patient cells. B: Refractory patient cells. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com].

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One of the CML patients exhibited a specific pattern. Before treatment, two populations with different levels of tyrosine phosphorylation were detected. Addition of imatinib significantly inhibited tyrosine hyperphosphorylation expression with an estimated IC50 of 1 μM. However, few cells remained hyperphosphorylated even with high doses of the inhibitor, suggesting that the cells were resistant (Fig. 8A). This patient was treated with imatinib, but relapsed a few months later. Analysis of phosphotyrosine at the time of relapse shows a single hyperphosphorylated population (Fig. 8B). The tyrosine hyperphosphorylation of this cell population decreased with imatinib treatment, but remained hyperphosphorylated as compared to sensitive cells (IC50 = 5 μM) (Fig. 8B). Therefore, the resistant population detected before treatment still persisted after imatinib treatment and was even predominantly present.

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Figure 8. Detection of imatinib resistance in one CML patient. Mononuclear cells from a resistant CML patient were stained with p-tyr antibody (PY99) after 2 h of treatment with imatinib (0–50 μM) at time of protocol inclusion (A) and at time of relapse (B). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com].

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Tyrosine Phosphorylation Study in Imatinib-Resistant ALL Patient Cells

Patients with Ph+ ALL are also treated with imatinib, but as in CML patients, drug resistance appears within a few months in most of them (32). For this reason, we investigated the level of tyrosine phosphorylation in mononuclear cells from Ph+ ALL patients. Two populations with different levels of phosphorylation were detected, i.e., hyperphosphorylated Bcr-Abl positive cells (blastic population) and nonphosphorylated Bcr-Abl negative (leukocytes) cells. Indeed in this sample, the blastic cells correspond to 75% of total cells (Fig. 9A). Addition of 1 μM of imatinib had no effect on the level of tyrosine phosphorylation in these two populations (Fig. 9A). Analysis of normal blood mononuclear cells using the same technique showed no hyperphosphorylation of these cells after staining (Fig. 9B).

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Figure 9. A: Effect of imatinib on tyrosine phosphorylation in ALL patients. Mononuclear cells from one Ph+ ALL sample were labeled with p-tyr antibody (PY99) after 2 h of treatment with imatinib (1 μM). B: Effect of imatinib on tyrosine phosphorylation in normal cells. Mononuclear cells from normal sample were labeled with p-tyr antibody and analyzed by flow cytometry.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

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).

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

We are grateful to Dr. Elizabeth Buchdunger (Novartis, Basel, Switzerland) for the generous gift of imatinib mesylate.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
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