Figure 5.

Inhibition of c-Abl kinase activity by imatinib in A431 cells and sensitivity to ADCC. A & B, A431 cells were seeded overnight in 96-well plates and treated with vehicle (0, DMSO) or imatinib (0.1, 1 or 10 μM) for 48 h. Treatments were aspirated and replaced with fresh growth media. Effector cells were added in the absence or presence of cetuximab (0.01, 0.1, and 1 μg/mL). Cytotoxicity was assessed 4 h later and specific lysis by effector cells was determined. For A, 20,000 NK92-CD16V effector cells were used. Results represent three independent experiments (n=3). For B, 50,000 IL-2 negatively-selected, IL2 stimulated NK effector cells were used. Percent-change in specific lysis was quantified to account for varying levels of donor specific lysis against target cells. Results represent one of two independent experiments using three independent donors (n=3). For A & B, imatinib pre-treatment was compared to vehicle within each sub-panel. *, p<0.05; **, p<0.01; and ***, p<0.001 by two-tailed t-test. C, A431 cells were seeded in six-well plates overnight and treated with vehicle (0, DMSO) or imatinib (0.1, 1 or 10 μM) and cell lysates collected 48 h later. Western blots were conducted and membranes blotted for phospho-CrkL (p-CrkL), stripped and re-blotted for both total CrkL and then β-actin. Densitometry was conducted and p-CrkL levels were normalized to total CrkL levels and then compared to vehicle treatment. Results are for one representative of two experiments. D, A431 cells were seeded overnight in 96-well plates and treated with vehicle (0, DMSO) or imatinib (0.1, 1 or 10 μM) for 48 h. Viability was assessed by fluorometric assay. No statistically significant differences were found between treatments by t-test. Results are from six independent experiments (n=6). For panels A, B & C, error bars represent s.d. of the mean.