Effect of amino acid substitutions at positions 315 and 380 on inhibition of Bcr-Abl by imatinib mesylate.

(A) Stick representations of imatinib mesylate in complex with Abl or mutants of Abl at position 315 to methionine and 380 to threonine. Mutants were introduced into the Abl structure by means of Swiss Prot Structure Viewer software and are based on previously published coordinates of the Abl/imatinib mesylate analog complex.8(B) Cos7 cells were transfected with either pApuro, Bcr-Ablwt, or the indicated mutants of Bcr-Abl at positions 315 and 380. Cells were either left untreated or incubated with 10 μM imatinib mesylate at 4 hours prior to lysis. Phosphorylation of Bcr-Abl was assessed by means of anti–phospho-Abl antibodies. Expression of Bcr-Abl was controlled by means of anti-Abl antibody. (C) Survival and proliferation of 32D cells expressing Bcr-Ablwt or the various mutants of Bcr-Abl at positions 315 and 380. First, 5 × 10⁴ cells were seeded per 6 wells and treated with DMSO, 1 or 2.5 μM imatinib mesylate. Survival and proliferation were determined 48 hours later as described in “Materials and methods.” Data are shown as means ± SD.