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

Abstract

Abstract. HL‐60 and MCF‐7 cells were treated with 0.15 μM 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 standing 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.