Comparison of methods based on annexin-V binding, DNA content or TUNEL for evaluating cell death in HL-60 and adherent MCF-7 cells.
HL-60 and MCF-7 cells were treated with 0.15 microM camptothecin (CPT) or with the solvent dimethylsulfoxide (DMSO) for the controls, for 2, 3 and 4 h or for 24, 48 and 72 h, respectively. The apoptotic index (AI) was then evaluated in parallel by the following flow cytometric methods: (1) double staining of unfixed cells with fluoresceinated annexin V and propidium iodide (PI), this after detachment by trypsinization in the case of MCF-7 cultures; (2) prefixation in 70% ethanol, extraction of degraded, low molecular weight DNA with 0.2 M phosphatecitrate buffer and analysis of the DNA content stained with PI; (3) TUNEL, i.e. labelling of DNA strand breaks with biotin-dUTP, followed by staining with streptavidin-fluorescein and counterstaining with PI. In HL-60 cells, the three methods gave similar results for the AI (3-4% in the controls and at 2 h of CPT treatment, and 35-43% at 3 and 4 h after CPT). This indicates that CPT-induced membrane alteration and DNA fragmentation occurred concomitantly in those cells. For MCF-7 cells, CPT-induced apoptosis developed more slowly, the AI, whether based on annexin V or on DNA content, remained unchanged at 24 h, then was increasing to 8% at 48 h and to 25% at 72 h of treatment. In these cells, the TUNEL index did not increase prior to 72 h, and the increase was minor (up to 9% vs. 2-3% in the controls) at 72 h of the treatment. This indicates that in MCF-7 cells DNA strand breaks cannot be effectively labelled, which may be due to inaccessibility of 3'-OH ends in the breaks to exogenous terminal deoxynucleotidyl transferase. The mechanism of endonucleolytic DNA fragmentation thus may be different, depending on the cell type.
Del Bino
G
G
Laboratory for Cytology and Experimental Oncology, Faculty of Medicine, Free University of Brussels (ULB), Belgium.
Darzynkiewicz
Z
Z
Degraef
C
C
Mosselmans
R
R
Fokan
D
D
Galand
P
P
D017304:Annexin A5 / Q000378:metabolism*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001943:Breast Neoplasms; D002166:Camptothecin / Q000494:pharmacology; D003584:Cytological Techniques; D053938:DNA Fragmentation; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D004273:DNA, Neoplasm / Q000032:analysis*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D018922:HL-60 Cells / Q000166:cytology* / Q000201:enzymology; D006801:Humans; D020287:In Situ Nick-End Labeling; D012997:Solvents / Q000494:pharmacology
D017304:Annexin A5; D000972:Antineoplastic Agents, Phytogenic; D004273:DNA, Neoplasm; D012997:Solvents; D004253:DNA Nucleotidylexotransferase; D002166:Camptothecin; D004121:Dimethyl Sulfoxide