Rituximab-induced apoptosis and p38 MAP-kinase activity.
(A) Freshly isolated B-CLL cells (2 × 106/mL) were cultured for the indicated time periods, in the presence or absence of 4 μg/mL rituximab with or without the cross-linking mouse anti–human IgG1F(ab)2 fragment (20 μg/mL). Cells were harvested, and whole cell lysates were quantified and loaded on 10% polyacrylamide gels. Following electrophoresis and blotting, the membranes were developed by means of antibodies specific for the phosphorylated forms of JNK, ERK, and p38 and by chemiluminiscence. Equal loading of the gels was confirmed by reprobing of the membranes with antibodies specific for total JNK, ERK, and p38. Shown is 1 representative experiment out of 3. (B) Freshly isolated B-CLL cells from 7 B-CLL patients were cultured (2 × 106/mL) in duplicates and preincubated for 1 hour with 10μM SB203580, an inhibitor of the p38 MAP-kinase pathway. Then, the B-CLL cells were cultured in the presence or absence of 4 μg/mL rituximab and 20 μg/mL mouse anti–human IgG1F(ab)2 fragment for 24 hours. For quantification of the degree of apoptosis, isolated nuclei from 5 × 105 were lysed, stained with PI, and analyzed for DNA content on a FACSort flow cytometer (left). To demonstrate inhibition of the p38 MAP-kinase pathway under these conditions, MAPKAP K2 in vitro kinase assays were carried out. B-CLL cells were preincubated with 10 μM SB203580 for 1 hour and stimulated as described above for 8 hours. The B-CLL cells were lysed and immunoprecipitated with specific MAPKAP K2 antibodies, and kinase assays were performed with the use of a specific MAPKAP K2 substrate (right; mean and SEM).
