Effect of MCD and filipin on ET-18-OCH3–induced apoptosis and Fas clustering in human leukemic cells.
(A) HL-60 cells were pretreated with MCD (15 μg/mL) for 1 hour at 37°C in serum-free medium, and then ET-18-OCH3 was added at 1 or 3 μg/mL for 5 hours. Cells were stained with propidium iodide, and their DNA content was analyzed by fluorescence flow cytometry. The percentage of cells with a DNA content less than G1 (sub-G1) is indicated in each histogram. Control untreated cells and cells treated only with MCD were run in parallel. Data are representative of 3 experiments performed. (B) HL-60 cells were pretreated with MCD (15 μg/mL) or filipin (1 μg/mL) for 1 hour at 37°C in serum-free medium, and then ET-18-OCH3was added at 1 (ET 1) or 3 (ET 3) μg/mL for 5 hours. Percentage of apoptotic cells was determined as above. Control untreated cells and cells treated only with MCD or filipin were also run in parallel. Data shown are means of 3 independent experiments ± SD (C). Bone marrow leukemic primary cultures derived from patients with M2 or M3 leukemia were incubated in the absence (Control) or in the presence of 3 μg/mL ET-18-OCH3, MCD (15 μg/mL), or MCD plus ET-18-OCH3 for 3 hours. Fragmented DNA was extracted and analyzed as described previously.10 14 Fragmented DNA from 1.5 × 106 cells was loaded in each lane. A 123-bp DNA ladder was used as standard (STD). (D) HL-60 cells were untreated or pretreated with MCD (15 μg/mL) for 1 hour at 37°C in serum-free medium, and then ET-18-OCH3 was added at 1 μg/mL for 3 hours. Fas aggregation was then analyzed by confocal microscopy as indicated in Figure 1, using anti-Fas mAb and CY3-conjugated antimouse antibody. Bar indicates 8 μm.
