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 (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-alpha or camptothecin (CPT) in the absence or constant presence of 10-50 microM 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-alpha 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-alpha or CPT. The rate of cell entry to apoptosis was subsequently 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 approximately 8% of cells per hour; the remaining cells were undergoing apoptosis at a rate of approximately 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 approximately 7% of cells per hour. Remaining cells were undergoing apoptosis at a rate of approximately 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.
Smolewski
Piotr
P
Brander Cancer Research Institute, New York Medical College, 19 Bradhurst Avenue, Suite 2400, Hawthorne, NY 10532, USA.
Grabarek
Jerzy
J
Lee
Brian W
BW
Johnson
Gary L
GL
Darzynkiewicz
Zbigniew
Z
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001681:Biological Assay; D002166:Camptothecin / Q000494:pharmacology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018922:HL-60 Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism; D016196:S Phase / Q000187:drug effects / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D011956:Receptors, Cell Surface; D014409:Tumor Necrosis Factor-alpha; D020169:Caspases; D002166:Camptothecin