Kinetics of HL-60 cell entry to apoptosis during treatment with TNF-alpha or camptothecin assayed by the stathmo-apoptosis method

Background: Duration of apoptosis, from onset to final disintegration of the cell, is often short and variable. The apoptotic index (M), 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) FM-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-alpha or camptothecin (CPT) in the absence or constant presence of 10-50 muM FLICA. The CAI was measured at different time points for up to 48 h by flow cytometre. 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-alpha or CPT alone was prevented in Cultures containing FLICA. Addition of FLICA alone had no effect on cell viability. The percentage of FLICA-labelcd cells was plotted as a function of time after addition of TNF-alpha. or CPT. The rate of cell entry to apoptosis was subsequently3, estimated from the slopes of the stathmo-apoptotic plot. The slopes revealed that the TNF-alpha or CPT-treated cells asynchronously underwent apoptosis with a stochastic-like kinetics and at two different rates. About 50% of cells in the TNF-alpha-treated cultures underwent apoptosis during the initial 6 h at a rate of similar to8%, of cells per hour the remaining cells were undergoing apoptosis at a rate of similar to2.59% 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 similar to(-)% of cells per hour. Remaining cells were undergoing apoptosis at a rate of similar to1% of cells per hour during up to 48 11 exposure to CPT. Spontaneous apoptosis in the untreated Cultures occurred at a rate of 0.29% 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. (C) 2002 Wiley-Liss, Inc.