Figure 4.

P-gp affects caspase-3-dependent DNA fragmentation in drug-resistant cell lines. CEM cell lines [CCRF (P-gp^low) and A7⁺ (P-gp^high)] were labeled with ¹²⁵IUdR for 1 h, washed in growth media, and incubated for 16–48 h in 96-well plates (2 × 10⁴ cells/well) with cell death stimuli at final concentrations as follows: (a) DOX (0.1 μg/ml, 48 h); (b) anti-human Fas IgM mAb CH-11 (0.01 μg/ml, 16 h). In some wells cells were preincubated for 30 min with anti-P-gp mAb (MRK-16) (50 μg/ml, final concentration) and/or followed by 20 μM (final concentration) ZDEVD-fmk or ZYVAD-fmk inhibitor for 30 min. Cell death stimuli then were added as indicated for 16–48 h. These data are calculated as the mean ± SE of duplicate samples and are representative of at least two different experiments. Protein lysates from CCRF (c) or A7⁺ (d) cells treated with anti-Fas IgM (0.01 μg/ml, 4 h) in the presence or absence of MRK-16 (50 μg/ml, final concentration) or verapamil (5 μM) were separated by SDS/PAGE, and Western blots were performed by using an anti-caspase-3 mAb. The addition of anti-Fas IgM, MRK-16, and verapamil is indicated by the table above the blots, and the position of molecular weight markers in kDa is indicated on the left. The expression of pro-caspase-3 and an active cleaved product is indicated by arrows.