Mechanisms of A3D8-mediated inhibition of apoptosis.

(A and B) Inhibitory effect of anti-CD44 A3D8 mAb on myeloid leukemia cell proliferation. HL60 (A) and HL60/Bcl-2 (B) cells in exponential growth phase were seeded at 5 × 10⁵/mL in culture medium in the absence (medium) or presence of 2.5 μg/mL anti-CD44 mAbs (A3D8 or J173). Cell viability was evaluated by trypan blue dye exclusion. (C) Inhibitory effect of A3D8 anti-CD44 mAb on DNR-induced ceramide generation in HL60 cells. Cellular lipids were metabolically labeled by incubating cells with [³H]-palmitic acid for 48 hours. A3D8 mAb or control medium was added during the last 16 hours of this incubation. DNR (0.5 μmol/L) was then added and the amount of cellular ceramide was quantitated through 15 minutes, as described.13 Results shown represent the mean ± SEM from 3 independent experiments. The difference between A3D8-treated and untreated cells is statistically significant (Student t test, P < .05). (D) Additive inhibitory effects of A3D8 anti-CD44 mAb and Bcl-2 on DNR-induced apoptosis. HL60/Bcl-2 (Bcl-2 overexpressing) and HL60/Neo (control) cells were preincubated for 16 hours with 2.5 μg/mL anti-CD44 mAb A3D8 or J173, or with culture medium only; cells were then treated with 0.5 μmol/L DNR for 24 hours, and the percentage of apoptotic cells was evaluated by microscope examination. Results represent the mean ± SEM from 3 independent experiments. (E) Western blot analysis of Bcl-2 expression in HL60/Neo and HL60/Bcl-2 cells.