Comparison of methods based on annexin-V binding, DNA content or TUNEL forevaluating cell death in HL-60 and adherent MCF-7 cells

HL-60 and MCF-7 cells were treated or with the solvent dimethylsulfoxide (D MSO) for the controls, for 2, 3 and 4 h or for 24, 48 and 72 h, respectivel y. The apoptotic index (AI) was then evaluated in parallel by the following flow cytometric methods: (I) double staining of unfixed cells with fluores ceinated annexin V and propidium iodide (PI), this after detachment by tryp sinization in the case of MCF-7 cultures; (2) prefixation in 70% ethanol, e xtraction of degraded, low molecular weight DNA with 0.2 hi phosphatecitrat e buffer and analysis of the DNA content stained with PI; (3) TUNEL, i.e. l abelling of DNA strand breaks with biotin-dUTP, followed by staining with s treptavidin-fluorescein and counterstaining with PI. In HL-60 cells, the th ree 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 concomitant ly in those cells. For MCF-7 cells, CPT-induced apoptosis developed more sl owly, the AI, whether based on annexin V or on DNA content, remained unchan ged at 24 h, then was increasing to 8% at 48 h and to 25% at 72 h of treatm ent. In these cells, the TUNEL index did not increase prior to 72 h, and th e increase was minor (up to 9% vs. 2-3% in the controls) at 72 h of the tre atment. This indicates that in MCF-7 cells DNA strand breaks cannot be effe ctively 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.