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

Abstract

An early sign of apoptosis in many cells is the appearance of phosphatidylserine (PS) on the outside of the plasma membrane, whilst the cells still retain the ability to exclude DNA-binding molecules such as propidium iodide and 7-aminoactinomycin 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 annexin V binds to camptothecin (CAM)-treated HL-60 cells, a commonly used model for apoptosis. We investigated the effects of culturing HL-60 cells for up to 8 h with a range of CAM concentrations. We used flow cytometry to measure cellular light scatter, annexin V-FITC binding, and 7-AAD uptake, and DNA content after fixation and permeabilization. We also used microscopy to examine the morphology of cells (both unsorted and sorted according to their light scatter) after cytocentrifugation. We found that CAM caused the rapid appearance of low 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 annexin V binding could be detected to both intact cells and to apoptotic bodies. When they first appeared, the intact annexin V+ cells had S-phase DNA content. During CAM-induced apoptosis of HL-60 cells, the external exposure of PS can either precede or follow DNA cleavage, which suggests that PS exposure is not always an indicator of early apoptosis.