Stathmo-apoptosis: Arresting apoptosis by fluorochrome-labeled inhibitor of caspases

Apoptosis, like mitosis, is a kinetic event. The entire duration of apoptosis, from its onset to total disintegration of the cell, is often short and may be of variable duration. The time-window through which individual apoptotic cells display their characteristic features that serve to identify them varies depending on: a) the assay that is used, b) the cell type, c) the nature of the inducer of apoptosis, and d) the environmental factors the cell is exposed to that may shorten or prolong apoptosis. Thus, because the apoptotic index (AI) does not accurately represent incidence of apoptosis it is desirable to estimate the rate of cell death in analogy to the cell birth rate which is assessed by the stathmo-kinetic approach by arresting cells in mitosis. In this study the fluorescent caspase inhibitor FAM-VAD-FMK was used for dual purposes: a) to arrest the process of apoptosis (stathmo-apoptosis), and b) to have the arrested cells labeled with fluorochrome. Apoptosis of HL-60 and MCF-7 cells was induced by DNA topoisomerase I inhibitor camptothecin (CPT) and FAM-VAD-FMK was added at the same time as the inducer. While the cells become progressively labeled with FAM-VAD-FMK, their disintegration, loss of the phase-contrast and loss of the capability to bind the inhibitor, and in the case of MCF-7 cells, detachment from the slides, all were prevented for up to 48 h. The percentage of FAM-VAD-FMK labeled HL-60 cells was plotted as a function of time after addition of CPT and the rate of cell entrance to apoptosis was estimated from the slopes of the stathmo-apoptotic plot at different time after administration of CPT. The plot revealed the presence of two distinct subpopulations: during the initial 8 h of the treatment with CPT the cells of the first subpopulation, predominantly the S-phase cells, were entering apoptosis at a rate of about 7% of cells per hour. The remaining cells were stochastically entering apoptosis between 8 and 48 h at a rate 1% of cells per hour. The present approach offers a unique capability to accurately estimate the kinetics of cell transition to apoptosis, revealing the unbiased cumulative apoptotic index over a long time span after induction of apoptosis.