Microarray analysis of genes downregulated by dexamethasone. Primary thymocytes were harvested from adult B6 mice, treated with vehicle (0.1% ethanol) or 10⁻⁶ M dexamethasone for 12 h, and subjected to microarray analysis as previously described (see Materials and Methods). The bar graph shows the extent of downregulation relative to vehicle-treated cells. Lck (designated with an asterisk) had a signal Log₂ ratio of (−) 2.6 indicating repression by a magnitude of sixfold. Lck was also downregulated in WEHI7.2 and S49A.2 murine T-cell lines

Dexamethasone downregulates Lck to inhibit TCR signaling. (a) Single cell calcium measurements in unstimulated WEHI7.2 cells treated with 10⁻⁸ M dexamethasone for 20 h. Representative traces are shown. (b) Cells were treated as in a, but stimulated with anti-CD3 antibody (2 μg/ml) 1 min into the calcium trace. (c) Quantification of oscillating cells (≥ three spikes) in a and b. (d) Cells were transfected with control (nontargeting) siRNAs or those that specifically target Lck and incubated for 24 h. Lysates were subjected to western blotting for Lck. (e) Representative calcium traces in nontargeting or Lck siRNA-transfected cells after stimulation with anti-CD3 (2 μg/ml). (f) Quantification of oscillating cells in e. Data are representative of multiple independent experiments. Error bars represent the S.E.M.

Dexamethasone and dasatinib synergistically inhibit TCR signaling. (a) WEHI7.2 cells were treated with vehicles (0.1% ethanol and 0.01% DMSO), 10⁻⁶ M dexamethasone, 100 nM dasatinib, or both for 24 h. Phospho-Lck (Y394 and Y505), total Lck, and Fyn levels were measured by western blotting. β-Actin was used as a loading control. (b) Quantification of phospho-Lck (Y394) after treatment with dexamethasone or dasatinib. (c) Cells were treated as in a and downstream TCR proteins ZAP-70, LAT, SLP-76, and phospho-MEK were measured by western blotting. Data are representative of multiple independent experiments. Error bars represent the S.E.M.

Aberrant Lck expression correlates with resistance to dexamethasone in CLL. (a) Representative single cell calcium traces in unstimulated PBLs from CLL2 showing that these cells undergo ligand-independent signaling. Ligand-independent calcium oscillations were detected in three of three CLL patient samples. (b) PBLs from CLL14 (2 × 10⁶ cells per ml) were treated with vehicle (0.1% ethanol) or 10⁻⁶ M dexamethasone for 24 – 96 h. (c) MEC1 cells (2 × 10⁵ cells per ml) were treated with vehicle or 10⁻⁶ M dexamethasone for 24 h. Cell viability was measured in triplicate by trypan blue dye exclusion. Similar results were obtained by MTS assay. (d) Lck mRNA was measured by real-time PCR in 10 freshly isolated primary CLL samples and in PBLs from a patient with circulating marginal zone lymphoma. Lck was also measured in the prolymphocytoid CLL line MEC1, primary thymocytes, WEHI7.2, CEMC7, and Jurkat cells. All values were measured in triplicate relative to normal B cells. β-Actin was used as an endogenous control for each PCR reaction. (e) Fyn and Lyn were measured as in d. Values represent the mean change in mRNA expression relative to normal B cells. Significance was determined by Student’s t-test. (f) Lck protein was measured by western blotting in six freshly isolated primary CLL samples. β-Actin was used as a loading control (lysate from CLL2 was not loaded in lane three due to insufficient sample volume). (g) A scatter-plot showing a significant negative correlation between Lck mRNA level and response to dexamethasone. Lck mRNA was measured as in d (x axis) and response to dexamethasone (y axis) was determined by calculating fold change in cell killing of CLL cells (10⁻⁶ M dex, 24 h). The scatter-plot shows the Spearman’s rank order assigned for each variable pair (N = 9). The correlation coefficient is − 0.700 (P<0.05). Data are representative of multiple independent experiments. Error bars indicate the difference between maximum and mean values from triplicate measurements or the S.E.M from real-time PCR and viability data, respectively.

Dexamethasone downregulates expression of Lck. (a) WEHI7.2 cells were treated with vehicle or varying concentrations of dexamethasone for 24 h. Lck mRNA was measured by real-time PCR. Values represent triplicate measurements and are relative to the vehicle-treated sample. β-actin was used as an endogenous control for each PCR reaction. (b) Primary thymocytes (5.0 × 10⁶ cells per ml) were harvested from adult B6 mice and treated with vehicle or dexamethasone for 12 and 24 h. Lck mRNA was measured by real-time PCR as described above. (c) WEHI7.2 cells were treated with vehicle or 10⁻⁶ M dexamethasone with and without the glucocorticoid receptor antagonist RU486 at equal and excess concentrations for 24 h. Lysates were subjected to western blotting for Lck. (d) Thymocytes were treated with vehicle or 10⁻⁸ M dexamethasone for 12 h and subjected to western blotting for Lck. (e) CEMC7 cells (T-ALL) were treated with varying concentrations of dexamethasone for 48 and 72 h and subjected to western blotting for Lck. β-Actin was used as a loading control. Data are representative of multiple independent experiments. Error bars indicate the difference between maximum and mean values from triplicate measurements

Dasatinib inhibits Lck phosphorylation and TCR signaling. (a) WEHI7.2 cells were treated with vehicle (0.01% DMSO) or dasatinib for 30 min. Phosphorylated (Y394) and total Lck levels were measured by western blotting. β-Actin was used as a loading control. (b) Single cell calcium measurements in WEHI7.2 cells treated with 100 nM dasatinib for 30 min and stimulated with anti-CD3 antibody (2 μg/ml) 1 min into the calcium trace. Representative traces are shown. (c) Quantification of oscillating cells (≥ three spikes) in b. (d) Cells were treated with dasatinib as in a, and phospho-MEK and ERK levels were measured by western blotting. Cells were stimulated with anti-CD3 (2 μg/ml) for 5 min to induce MEK and ERK phosphorylation. Data are representative of multiple independent experiments. Error bars represent the S.E.M.

Lck inhibition enhances glucocorticoid sensitivity and apoptosis. (a) WEHI7.2 cells were treated with vehicles (0.1% ethanol and 0.01% DMSO), 10⁻⁶ M dexamethasone, 100 nM dasatinib, or both for 24 h. Cells were transferred onto a 96-well plate, treated with MTS reagent, and incubated for 3 h. Values represent the percent of control (Abs at 490 nm) and were obtained in triplicate. Inset, IC₅₀ for dexamethasone treatment alone or dexamethasone and dasatinib. (b) Cells were treated as in a and apoptosis was measured by flow cytometric analysis of Annexin V-positive cells. (c) Cells were treated as in a, lysed in non-denaturing buffer, and Caspase-3 activity was assessed by adding Ac-DEVD-AMC fluorogenic substrate. Values represent fold increase in fluorescence and were obtained in triplicate. (d) Cells were treated as in a, stained with Hoechst 33342, and visualized by epifluorescence microscopy (Zeiss axiovert S100) using a Zeiss × 40 fluorescent oil objective. Representative fields are shown for each treatment. Apoptotic nuclear morphology is indicated by white arrows. (e) WEHI7.2 cells were transduced with lentiviral shRNAs to selectively target Lck. Top: western blot for Lck and Fyn in cells transduced with empty vector or Lck shRNA. Bottom: control or Lck knockdown cells (2.0 × 10⁵ cells per ml) were treated with vehicle (0.1% ethanol) or dexamethasone for 24 h. Apoptosis was measured as in b. Data are representative of multiple independent experiments. Error bars represent the S.E.M.

Dasatinib enhances glucocorticoid sensitivity and apoptosis in primary CLL cells. (a and b) PBLs from CLL2 were treated with vehicle (0.1% ethanol) or 10⁻⁶ M dexamethasone or with vehicles (0.1% ethanol and 0.01% DMSO), dexamethasone, 100 nM dasatinib or both for 24 h. Total Lck and phospho-Lck (Y394) were measured by western blotting (note that the antibody used to detect phospho-Lck may also crossreact with phospho-Lyn). β-Actin was used as a loading control. (c) PBLs from CLL2 were treated with vehicle or dexamethasone (10⁻⁶ M) for 3, 6, and 24 h and GR-responsive protein Txnip was measured by western blotting to control for glucocorticoid uptake and GR functionality. (d) CLL cells and PBLs from a patient with leukemic mantle cell lymphoma were incubated in media containing vehicles (0.1% ethanol and 0.01% DMSO), dexamethasone, 100 nM dasatinib, or both for 24 h. Values represent the mean from treated and control samples. Statistical significance was determined by Student’s t-test. (e) PBLs from CLL14 were treated as in d with 10⁻⁶ M dexamethasone for 72 h. (f) MEC1 cells were treated as in d for 24 h. (g) PBLs from CLL6 were treated as in d with 10⁻⁶ M dexamethasone for 24 h. Apoptosis was determined by flow cytometric analysis of Annexin V and propidium iodide-stained cells. Control experiments are represented in blue dots. Experimental treatments are overlaid in red dots. (h) PBLs from CLL23 were treated with 10⁻⁶ M dexamethasone and 10 μM PP2 for 72 h. (i) PBLs from CLL5 were treated with vehicles (0.1% ethanol and 0.005% DMSO), 10⁻⁶ M dexamethasone, 50 nM BIBF 1120, or both for 24 h. Cells were also treated with 0.1% ethanol or 10⁻⁶ M dexamethasone in the presence of either nonphosphorylated (control) EGQYEEIP or phosphorylated EGQY*EEIP H₂O soluble peptides (200 nM) for 24 h. Cell death was measured in triplicate by trypan blue dye exclusion. (e) Data are representative of multiple independent experiments. Error bars represent the S.E.M.