Real-Time Analysis of Imatinib- and Dasatinib-Induced Effects on Chronic Myelogenous Leukemia Cell Interaction with Fibronectin

<div><p>Attachment of stem leukemic cells to the bone marrow extracellular matrix increases their resistance to chemotherapy and contributes to the disease persistence. In chronic myelogenous leukemia (CML), the activity of the fusion BCR-ABL kinase affects adhesion signaling. Using real-time monitoring of microimpedance, we studied in detail the kinetics of interaction of human CML cells (JURL-MK1, MOLM-7) and of control BCR-ABL-negative leukemia cells (HEL, JURKAT) with fibronectin-coated surface. The effect of two clinically used kinase inhibitors, imatinib (a relatively specific c-ABL inhibitor) and dasatinib (dual ABL/SRC family kinase inhibitor), on cell binding to fibronectin is described. Both imatinib and low-dose (several nM) dasatinib reinforced CML cell interaction with fibronectin while no significant change was induced in BCR-ABL-negative cells. On the other hand, clinically relevant doses of dasatinib (100 nM) had almost no effect in CML cells. The efficiency of the inhibitors in blocking the activity of BCR-ABL and SRC-family kinases was assessed from the extent of phosphorylation at autophosphorylation sites. In both CML cell lines, SRC kinases were found to be transactivated by BCR-ABL. In the intracellular context, EC50 for BCR-ABL inhibition was in subnanomolar range for dasatinib and in submicromolar one for imatinib. EC50 for direct inhibition of LYN kinase was found to be about 20 nM for dasatinib and more than 10 µM for imatinib. Cells pretreated with 100 nM dasatinib were still able to bind to fibronectin and SRC kinases are thus not necessary for the formation of cell-matrix contacts. However, a minimal activity of SRC kinases might be required to mediate the increase in cell adhesivity induced by BCR-ABL inhibition. Indeed, active (autophosphorylated) LYN was found to localize in cell adhesive structures which were visualized using interference reflection microscopy.</p></div

Changes in cell interaction with fibronectin after inhibitor treatment.

<p>The cells (6×10⁴ per well) were seeded into fibronectin-coated E-plates. After the microimpedance signal stabilization, the appropriate inhibitor was added in triplets. Black circles: control cells. Time of inhibitor addition is indicated by an arrow. Microimpedance signal (cell index) was normalized to 1 at the time of inhibitor addition. The graphs show mean and standard deviation of well triplets. A,C,E: JURL-MK1 cells, B,D,F: MOLM-7 cells. A,B: imatinib was added at 1 µM (blue circles) or 10 µM (red squares) final concentration. C,D: dasatinib was added at 2 nM (blue circles) or 10 nM (red squares) final concentration. E,F: dasatinib was added at 100 nM final concentration (red circles).</p

Changes in the adhered cell fraction induced by dasatinib pretreatment.

<p>JURL-MK1 (A) or MOLM-7 (B) cells were pretreated for 30 min with dasatinib at the indicated concentrations, seeded into FN-coated wells and incubated for 1 h at 37°C. Thereafter, the plate was washed and the fraction of attached cells was determined fluorimetrically and normalized using the value found in the control sample. The graphs show the means and standard deviations from at least 4 experiments for each condition, the statistical significance of the observed differences was evaluated using Student's paired t-test. Results significantly differing from untreated controls are denoted by asterisks (* p<0.05, ** p<0.01).</p

Microscopic analysis of SRC kinases localization in cells on fibronectin.

<p>MOLM-7 (A, C, D) or JURL-MK1 (B) cells were plated on fibronectin-coated slide, incubated for 1 h at 37°C, fixed and stained with antibodies against phospho-SFK (A, B), LCK (C) or LYN (D). A, B: Immunofluorescence images (left) were taken from the slice immediately adjacent to the FN-coated surface. Right images in A and B were captured in interference reflection mode. Green signal: SFK, red: actin (stained with phalloidin), blue: cell nuclei (DAPI). Scale bars: 10 µm.</p

Dasatinib-induced cell death in BCR-ABL-positive and -negative cell lines.

<p>JURL-MK1 (full squares) and JURKAT (empty squares) cells were treated with dasatinib at different concentrations as indicated. After 45–48 h incubation, the fraction of non-viable cells was determined by counting of trypan blue-stained samples. Summary of 3 independent experiments for each cell line is shown.</p

Time-course of BCR-ABL and SFK dephosphorylation after treatment with inhibitors.

<p>Time-resolved decrease in the phosphorylation status of BCR-ABL and SFK in JURL-MK1 cells after treatment with 1 µM imatinib (A) or 2 nM dasatnib (B). The experiment was repeated with closely similar results.</p

Imatinib-induced dephosphorylation of SFK in BCR-ABL-positive and -negative cell lines.

<p>Phosphorylation status of SFK after 2 h incubation with imatinib was assessed by western-blotting. Summary of independent experiments for JURL- MK1 (A), MOLM-7 (B) and HEL cells (C).</p

EC50 values for effects induced by imatinib and dasatinib.

<p>Cells were treated with the inhibitors at different concentrations for 48 h and counted. Cell viability was assessed using Trypan blue exclusion test. Measured values were fitted by sigmoidal curves using GraphPad Prism 5.0 software. Given EC50 values for inhibition of cell growth and for cell death induction are means and standard deviations from at least 3 independent experiments.</p><p>EC50 values for effects induced by imatinib and dasatinib.</p

Dasatinib-induced dephosphorylation of SFK and BCR-ABL.

<p>Phosphorylation status of SFK and BCR-ABL after 2 h incubation with dasatinib. Quantification summary from 3 independent experiments and representative western-blots are shown for each cell line. A: JURL-MK1, B: MOLM-7, C: HEL, D: JURKAT.</p

SRC family kinase profiles of leukemic cell lines.

<p>A: p-Y416 SFK, LYN, HCK and LCK expression pattern in the used cell lines. B: Summary of relative expression levels of p-Y416 SFK, LYN, HCK and LCK kinases in cell lines. Several (2–5) western-blots were quantified and normalized to the signal of JURL-MK1 cell line (p-SFK, LYN), JURKAT cell line (HCK) or MOLM-7 cell line (LCK).</p