CD37-SMIP induces caspase-independent cell death in B-CLL cells. Freshly isolated B cells from 5 patients with CLL were treated with cross-linker alone (α-Fc), trastuzumab + α-Fc, CD37-SMIP + α-Fc, or fludarabine in the presence or absence of the pan-caspase inhibitor Z-VAD-fmk (150 μmol/L). (A) Z-VAD-fmk failed to inhibit CD37-SMIP–induced cell death. Percentage cytotoxicity shown represents Annexin V⁺ and/or PI⁺ cells normalized with the media control. Error bars represent SD among 5 B-CLL patient cell samples. (B) CD37-SMIP failed to induce activation of caspases in CLL cells. Cell lysates from primary CLL B cells treated with indicated conditions for 24 hours were assessed by Western blotting to detect PARP and caspase-3 cleavage. UV-irradiated Jurkat cell lysate was used as a positive control for both PARP and caspase 3 cleavage. (C) CD37-SMIP–induced tyrosine phosphorylation of cellular proteins. CLL cells were treated with indicated concentrations of CD37-SMIP with or without cross-linker in PBS for 10 minutes, and phosphotyrosine proteins were detected by Western blot analysis using anti-phosphotyrosine antibody 4G10. (D) Effect of tyrosine kinase inhibitor herbimycin on CD37-SMIP–induced tyrosine phosphorylation. Primary CLL cells were pretreated with media or herbimycin (3 μmol/L) before stimulation with CD37 SMIP + α-Fc. The tyrosine-phosphorylated proteins were detected by Western blot using anti-phosphotyrosine antibody 4G10. (E) Herbimycin inhibited CD37-SMIP–induced cell death in primary CLL cells. CLL B cells were treated with indicated concentrations of herbimycin followed by CD37-SMIP (5 μg/mL) + α-Fc. The cellular cytotoxicity was measured by FITC-Annexin V/PI staining at 48 hours after treatment. Data shown represents percentage inhibition of CD37-SMIP–induced cytotoxicity in the presence of indicated concentrations of herbimycin. Results are summary of 11 CLL patient samples. Error bars are the SD among these samples.