Effects of FLIP antisense and TRAIL on CLL B cells.

(A) Antisense (AS) FLIP or random (R) control oligonucleotides (300 nM) were introduced into CLL B cells by electroporation. CLL B cells were then cultured overnight in the presence or absence of 100 nM TRAIL. The CLL B cells were recovered from cultures, and the percentage of apoptosis was determined by annexin V–FITC/PI double staining by means of flow cytometric analysis (bottom). Simultaneously, protein-containing lysates were prepared, normalized for total protein content (12.5 μg per lane), and analyzed by SDS-PAGE/immunoblot assay with the use of antibodies specific for FLIP or β-actin (top). (Data are representative of 3 independent experiments.) (B) Freshly isolated CLL B cells were cultured in the presence or absence of 1 μM CDDO. After 6 hours, 100 nM TRAIL was added, and the CLL B cells were cultured overnight. The percentage of apoptosis was determined by double staining with annexin V–FITC and PI with the use of flow cytometric analysis. Shown are the CLL patient specimens in which CDDO by itself induced less than 50% apoptosis (n = 5, of a total of 30).