Antiapoptotic gene and protein profile of CLL induced by CD40 stimulation is reversed by kinase inhibitors imatinib and dasatinib. (A) CLL cells were cocultured with control 3T3 or CD40L-expressing cells for 48 hours, in the presence of PD98059, imatinib, or dasatinib as indicated. Lysates were probed for Bim, Mcl-1, Bcl-XL, A1/Bfl-1, and Bcl-2 as indicated and actin as loading control. Shown are representative examples of 2 CLL samples with wild-type (WT) p53 function (left and middle panels), and 1 CLL with p53 dysfunction (right panel). Note different order of samples in this panel and that the lanes of the A1/Bfl1 blot have been repositioned to match the other blots from the same experiment. Vertical lines have been inserted to mark the adjusted lanes. The up-regulation of Mcl-1, Bcl-XL, and A1/Bfl-1 is not affected by ERK inhibition, but prevented by imatinib or dasatinib, irrespective of p53 functionality. (B) RNA was collected from CLL cells stimulated for 48 hours with CD40 and inhibitors as indicated, and assayed for expression of 34 apoptosis genes by MLPA. Shown are averaged relative expression levels plus or minus SD (in percentage of total normalized signal) of selected genes in samples from p53 WT (n = 4) and p53 dysfunctional (n = 3) CLL cells. The CD40-mediated positive effects on transcription of A1/Bfl-1 and Bcl-XL are reversed by Abl kinase inhibitors. Examples of genes that are not significantly affected at the transciptional level are Mcl-1, Bim, and GUS (β-glucuronidase, a housekeeping gene).