Ethanol induced apoptosis in human HL-60 cells

In this study flow cytometric and morphologic methods of apoptosis detectio n in human promyelocytic leukemia cell line HL-60 were compared. HL-60 cell s were harvested at 4, 7, 16, 24 a 48 hours after induction of apoptosis by 3 % ethanol. Little changes were observed both by flow cytometry (decrease of forward scatter, increase of unprocessed cells staining with APO2.7 ant ibody) and viability determination by Trypan-blue staining until after 7 ho urs. However, after 4 hours morphologic changes were observed in the nuclea r and cytoplasmic structures using Diff-Quik stained cytospin preparations and standard light microscopic techniques (50% apoptotic cells). The same r esults were obtained by flow cytometric measurement of sub-diploid DNA cont ent (sub-G(1) cells), and an increase of staining with APO2.7 antibody in c ells permeabilised by digitonin prior to staining. After 7 hours almost all cells exhibited apoptotic morphology. After 16 hours the cell size (forwar d scatter) decreased significantly, and 54% of unprocessed cells were APO2. 7 positive. After 24 hours only 6% of cells were alive thigh forward scatte r) and these cells were APO2.7 negative. The HL-60 cells did not proliferat e during the cultivation in 3% ethanol, and after 48 hours all stained by T rypan blue. HL-60 leukemic cells were CD34(-)/AC133(-), CD33(+)/CD15(+), an d only 2% of the cells were CD95(+). Induction of apoptosis by ethanol did not enhance CD95 antigen expression.