Assay of caspase activation in situ combined with probing plasma membrane integrity to detect three distinct stages of apoptosis.

Activation of cysteine-aspartic acid specific proteases (caspases) in situ, in live cells, can be detected using fluorochrome-labeled inhibitors of caspases (FLICA), the reagents that covalently bind to the active center of these enzymes. In the present study, this assay was combined with a probe of plasma membrane capacity to exclude the cationic fluorochrome propidium iodide (PI). Apoptosis of HL-60 cells was induced by DNA topoisomerase I inhibitor camptothecin (CPT). The cells were then incubated with FAM-VAD-fluoro-methyl ketone (FMK), the pan-caspase FLICA, and subsequently briefly exposed to PI. The intensity of cellular green fluorescence of FLICA and red fluorescence of PI was measured by laser scanning cytometry (LSC) as well as by flow cytometry. Four distinct subpopulations were distinguished based on differences in fluorescence intensity. The subpopulations represented the sequential transitions from the stage when (a) the cells were both FLICA and PI negative (FLICA-/PI-), through the stages when (b) their caspases become progressively activated (FLICA+/PI-), (c) when their plasma membrane ability to exclude PI was lost (FLICA+/PI+), and finally (d) when the cell propensity to bind FLICA was eliminated (FLICA-/PI+). By estimating the percentage of cells in each subpopulation at different time points after administration of CPT, it was possible to study the kinetics of the transitions. The cell entry to-and progression through-these substages was asynchronous. Following this "supravital" analysis, the cells may be fixed, permeabilized, their DNA stoichiometrically stained with PI and cell cycle distribution of each of the four subpopulations analyzed. The loss of cells' ability to bind FLICA at the late stage of apoptosis indicates that caspases are either inactivated, degraded or excreted at that time point. Hence, the late apoptotic cells may not be identified solely on the evidence of the presence of activated caspases. The direct transition from FLICA-/PI- to FLICA-/PI+, bypassing the FLICA+ stages, may be considered as the marker of a primary cell necrosis.