There is substantial nuclear and cellular disintegration before detectablephosphatidylserine exposure during the camptothecin-induced apoptosis of HL-60 cells

Background: An early sign of apoptosis in many cells is the appearance of p hosphatidylserine (PS) on the outside of the plasma membrane, whilst the ce lls still retain the ability to exclude DNA-binding molecules such as propi dium iodide and 7-aminoactinom) cin D (7-AAD). The protein annexin V binds preferentially to PS and has often been used to monitor the early phase of apoptosis. There have been some conflicting results concerning whether anne xin V binds to camptothecin (CAM)-treated HL-60 cells, a commonly used mode l for apoptosis. We investigated the effects of culturing HL-60 cells for u p to 8 h with a range of CAM concentrations. Methods: We used flow cytometry to measure cellular light scatter, annexin V-FITC binding, and 7-AAD uptake, and DNA content after fixation and permea bilization. We also used microscopy to examine the morphology of cells (bot h unsorted and sorted according to their light scatter) after cytocentrifug ation. Results: We found that CAM caused the rapid appearance of lon light scatter apoptotic bodies. Even among cells with "normal" light scatter, there was widespread DNA cleavage and nuclear fragmentation by 3 h. The percentage of apoptotic bodies peaked at about 4 h and it was only afterward that annexi n V binding could be detected to both intact cells and to apoptotic bodies. When they first appeared, the intact annexin Vf cells had S-phase DNA cont ent. Conclusions: During CAM-induced apoptosis of HL-60 cells, the external expo sure of PS can either precede or follow DNA cleavage, which suggests that P S exposure is not always an indicator of early apoptosis. Cytometry 40: 10- 18, 2000. (C) 2000 Wiley-Liss, Inc.