Figure 2.

Apoptosis induction by GD2-specific mAb 3F8 in human melanoma cell lines. (A) HTB63 cells were seeded in flat bottom six-well plates (2 × 10⁵/well) and incubated with mAb 3F8 (50 μg/ml). mAb HO-1 was used as an isotype matched control (Ctrl). Following a 48 h incubation at 37°C in a 5% CO₂ atmosphere, apoptosis induction was determined by Annexin V/7-AAD staining. Percentage of cells in different phases of apoptosis is indicated. (B) Colo38, HTB63, M21 and Melur cells were incubated with mAb 3F8 (50 μg/ml). mAb HO-1 was used as an isotype matched control (Ctrl). Following a 24 h incubation at 37°C in a 5% CO₂ atmosphere, apoptosis induction was determined by PI staining. A representative result obtained in HTB63 cells is shown (upper panel). Percentage of apoptotic cells detected as sub-diploid cells (SubG1 population) was correlated with expression levels of GD2 in the cell lines tested (lower panel). R² value as determined by the two order polynomial regression is indicated. The results presented are representative of those obtained in three independent experiments. (C) HTB63 cells were incubated with mAb 3F8 (50 μg/ml). mAb HO-1 was used as an isotype matched control (Ctrl). Untreated cells (Medium) and Jurkat cells treated with etoposide were used as a background and as a positive control for caspase 9 induction (Ctrl +), respectively. Following an incubation at 37°C in a 5% CO₂ atmosphere for the indicated times, cells were harvested and lysed. Cell lysates were analyzed by protein gel blot with the indicated mAbs. Calnexin was used as a loading control. The data shown are representative of the results obtained in three independent experiments. (D) HTB63 and M21 cells were incubated with mAb 3F8 (3F8) (50 μg/ml). Following an up to 24 h incubation at 37°C in a 5% CO₂ atmosphere enzymatic activity of activated caspase 3/7 in the cells was measured by Apo-ONE® Homogeneous Caspase 3/7 Assay. Data are expressed as mean ± SD of the results obtained in three independent experiments. (E) HTB63 cells were pre-incubated with 40 μM of the Pan-caspase inhibitor Boc-D-FMK, caspase 3 inhibitor Z-DQMD-FMK, caspase 8 inhibitor Z-IETD-FMK or caspase 9 inhibitor Z-LEHD-FMK. Following a 60 min incubation at 37°C in a 5% CO₂ atmosphere cells were then incubated with mAb 3F8 (50 μg/ml). Following a 24 h incubation at 37°C in a 5% CO₂ atmosphere apoptosis induction was determined by PI staining. Percentages of apoptotic cells detected as sub-diploid cells (SubG1 population) are shown. Data are expressed as mean ± SD of the results obtained in three independent experiments.