GA101-induced cell death is independent of DNA fragmentation, caspase activation, and BCL-2 overexpression. (A) Raji cells were treated with anti-CD20 mAb (10 μg/mL) or staurosporine (STSP, 2μM), a positive control for apoptosis, for 24 hours and DNA fragmentation was assessed using TUNEL staining analyzed by flow cytometry. Mean ± SEM of 3 independent experiments is shown on the left, with representative plots on the right. GA101 does not induce significant DNA fragmentation. (B) Wild-type Raji (left) and Raji cells that overexpress the antiapoptotic protein BCL-2, (Raji-BCL2; right), were preincubated with DMSO or Q-VD-OPH (20μM) for 30 minutes, then treated with anti-CD20 mAbs (10 μg/mL) or mitoxantrone (1 μg/mL), and cell death measured 48 hours later. Mean + SEM of 4 independent experiments are shown. Neither BCL-2 overexpression, caspase inhibition, nor a combination of both, had any impact on cell death induced by GA101, despite inhibiting mitoxantrone-induced apoptosis (P < .008). (C) Raji and Raji-BCL2 cells were treated as in panel B and growth inhibition assessed using the XTT assay as described in “Cell death and cell viability assays” 48 hours after treatment, with absorbance normalized relative to untreated cells. Mean + SEM of 4 independent experiments are shown. Caspase inhibition and BCL-2 overexpression had no impact on GA101-induced growth inhibition, both of which significantly attenuated growth inhibition induced by chemotherapy (mitoxantrone and doxorubicin, 1 μg/mL; P < .01). (D) Raji cells were treated with anti-CD20 mAbs or mitoxantrone as described above and Western blot analysis was performed for cleaved caspase 3 (CC3). No CC3 was observed following anti-CD20 mAb treatment (Ctl indicates control mAb; GA, GA101; Rit, rituximab; and Mtx, mitoxantrone).