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

Apoptosis, like mitosis, is a kinetic event. The entire duration of apoptos is, from its onset to total disintegration of the cell, is often short and may be of variable duration. The time-window through which individual apopt otic cells display their characteristic features that serve to identify the m 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 ce ll is exposed to that may shorten or prolong apoptosis. Thus, because the a poptotic index (AI) does not accurately represent incidence of apoptosis it is desirable to estimate the rate of cell death in analogy to the cell bir th rate which is assessed by the stathmo-kinetic approach by arresting cell s in mitosis. In this study the fluorescent caspase inhibitor FAM-VAD-FMK w as used for dual purposes: a) to arrest the process of apoptosis (stathmo-a poptosis), and b) to have the arrested cells labeled with fluorochrome. Apo ptosis of HL-60 and MCF-7 cells was induced by DNA topoisomerase I inhibito r camptothecin (CPT) and FAM-VAD-FMK was added at the same time as the indu cer. While the cells become progressively labeled with FAM-VAD-FMK, their d isintegration, loss of the phase-contrast and loss of the capability to bin d 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 st athmo-apoptotic plot at different time after administration of CPT. The plo t revealed the presence of two distinct subpopulations: during the initial 8 h of the treatment with CPT the cells of the first subpopulation, predomi nantly 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 off ers a unique capability to accurately estimate the kinetics of cell transit ion to apoptosis, revealing the unbiased cumulative apoptotic index over a long time span after induction of apoptosis.