There is substantial nuclear and cellular disintegration before detectable phosphatidylserine exposure during the
M A King1, M A Radicchi-Mastroianni, J V Wells
1Department of Clinical Immunology, Pacific Laboratory Medical Services, Royal North Shore Hospital, St. Leonards, New South Wales, Australia. making@doh.health.nsw.gov.au
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Insights
Phosphatidylserine (PS) exposure during camptothecin (CAM)-induced apoptosis in HL-60 cells does not always indicate early apoptosis, as it can occur before or after DNA cleavage. This challenges its use as a sole early apoptosis marker.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Phosphatidylserine (PS) externalization is a hallmark of early apoptosis, preceding loss of plasma membrane integrity.
- Annexin V is commonly used to detect PS exposure and monitor early apoptosis.
- Conflicting data exist regarding Annexin V binding to camptothecin (CAM)-treated HL-60 cells, a standard apoptosis model.
Purpose of the Study:
- To investigate the temporal relationship between PS exposure and DNA cleavage in CAM-induced apoptosis of HL-60 cells.
- To clarify the utility of Annexin V as an early apoptosis marker in this model.
Main Methods:
- Flow cytometry was employed to analyze light scatter, Annexin V-FITC binding, 7-aminoactinomycin D (7-AAD) uptake, and DNA content.
- Microscopy was used to assess cell morphology, including sorted populations based on light scatter.
Main Results:
- CAM induced rapid formation of low-light scatter apoptotic bodies.
- Widespread DNA cleavage and nuclear fragmentation were observed within 3 hours, even in cells with normal light scatter.
- Annexin V binding to intact cells and apoptotic bodies was detected primarily after 4 hours, following the peak of apoptotic body formation.
- Intact Annexin V-positive cells initially exhibited S-phase DNA content.
Conclusions:
- In CAM-induced HL-60 cell apoptosis, phosphatidylserine exposure can occur independently of DNA cleavage timing.
- This finding suggests that PS exposure is not a universally reliable indicator of early apoptosis in all contexts.
- The temporal dynamics of PS exposure and DNA fragmentation require careful consideration when assessing apoptosis induction.
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