Ethanol induced apoptosis in human HL-60 cells.
In this study flow cytometric and morphologic methods of apoptosis detection in human promyelocytic leukemia cell line HL-60 were compared. HL-60 cells 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 antibody) and viability determination by Trypan-blue staining until after 7 hours. However, after 4 hours morphologic changes were observed in the nuclear and cytoplasmic structures using Diff-Quik stained cytospin preparations and standard light microscopic techniques (50% apoptotic cells). The same results were obtained by flow cytometric measurement of sub-diploid DNA content (sub-G1 cells), and an increase of staining with APO2.7 antibody in cells permeabilised by digitonin prior to staining. After 7 hours almost all cells exhibited apoptotic morphology. After 16 hours the cell size (forward scatter) decreased significantly, and 54% of unprocessed cells were APO2.7 positive. After 24 hours only 6% of cells were alive (high forward scatter) and these cells were APO2.7 negative. The HL-60 cells did not proliferate during the cultivation in 3% ethanol, and after 48 hours all stained by Trypan blue. HL-60 leukemic cells were CD34-/AC133-, CD33+/CD15+, and only 2% of the cells were CD95+. Induction of apoptosis by ethanol did not enhance CD95 antigen expression.
Mareková
M
M
Department of Medical Biochemistry, Faculty of Medicine Hradec Králové, Charles University Prague, Czech Republic. marekova@lfhk.cuni.cz
Vávrová
J
J
Vokurková
D
D
D000071916:AC133 Antigen; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015703:Antigens, CD / Q000096:biosynthesis; D018952:Antigens, CD34 / Q000096:biosynthesis; D015214:Antigens, Differentiation, Myelomonocytic / Q000096:biosynthesis; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003593:Cytoplasm / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D005434:Flow Cytometry; D006023:Glycoproteins / Q000096:biosynthesis; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D016256:Lewis X Antigen / Q000096:biosynthesis; D010455:Peptides; D063268:Sialic Acid Binding Ig-like Lectin 3; D013997:Time Factors; D019014:fas Receptor / Q000096:biosynthesis
D000071916:AC133 Antigen; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018952:Antigens, CD34; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D006023:Glycoproteins; D016256:Lewis X Antigen; C000606023:PROM1 protein, human; D010455:Peptides; D063268:Sialic Acid Binding Ig-like Lectin 3; D019014:fas Receptor; D000431:Ethanol; D004247:DNA