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Keywords:

  • FLICA;
  • caspases;
  • cell necrobiology;
  • cell cycle;
  • S phase;
  • cumulative apoptotic index;
  • stathmo-apoptosis

Abstract

Background

Duration of apoptosis, from onset to final disintegration of the cell, is often short and variable. The apoptotic index (AI), as a snapshot of a transient event of variable length, does not truly represent incidence of apoptosis in the studied cell population. We recently proposed to estimate the cumulative apoptotic index (CAI) by inducing stathmo-apoptosis. A fluorescent inhibitor of caspases (FLICA) FAM-VAD-FMK is used to arrest the process of apoptosis and thereby prevent cell disintegration. Simultaneously, the arrested/apoptotic cells become FLICA-labeled. In the present study, this approach was applied to measure kinetics of HL-60 cell entrance into apoptosis induced via cell surface death receptor or a mitochondria-initiated pathway.

Materials and Methods

Cultures of HL-60 cells were treated with either TNF-α or camptothecin (CPT) in the absence or constant presence of 10–50 μM FLICA. The CAI was measured at different time points for up to 48 h by flow cytometry. Bivariate analysis of DNA content and cell labeling with FLICA was used to correlate apoptosis with the cell-cycle position.

Results

Selective loss of apoptotic cells seen in HL-60 cell cultures exposed to either TNF-α or CPT alone was prevented in cultures containing FLICA. Addition of FLICA alone had no effect on cell viability. The percentage of FLICA-labeled cells was plotted as a function of time after addition of TNF-α or CPT. The rate of cell entry to apop- tosis was subsequently estimated from the slopes of the stathmo-apoptotic plot. The slopes revealed that the TNF-α or CPT-treated cells asynchronously underwent apoptosis with a stochastic-like kinetics and at two different rates. About 50% of cells in the TNF-α-treated cultures underwent apoptosis during the initial 6 h at a rate of ∼8% of cells per hour; the remaining cells were undergoing apoptosis at a rate of ∼2.5% of cells per hour for up to 24 h. Also, about 50% of the CPT-treated cells, predominantly those in S phase of the cell cycle, underwent apoptosis within the initial 8 h of CPT exposure, at a rate of ∼7% of cells per hour. Remaining cells were undergoing apoptosis at a rate of ∼1% of cells per hour during up to 48 h exposure to CPT. Spontaneous apoptosis in the untreated cultures occurred at a rate of 0.2% of cells per hour.

Conclusions

This approach provides a means for measuring the kinetics of cell entrance to apoptosis (caspase activation) in large populations of cells in relation to the cell-cycle position. Cytometry 47:143–149, 2002. © 2002 Wiley-Liss, Inc.