Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor

There is evidence in the literature that serine (Ser) proteases, like caspases, are activated during apoptosis. Little is known, however, about individual Ser proteases and the mechanism of their activation. In the present study, we employed a new type of cell permeant reagent to detect activation of chymotrypsin-like proteases in human leukemic HL-60 cells induced to undergo apoptosis. The reagent, 5(6)-carboxyfluoresceinyl-L-phenylalanyl-chloromethyl ketone (FFCK), is a fluorochrome-labeled analog of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), the inhibitor known to specifically and covalently bind to the active center of chymotrypsin-like enzymes. In cultures treated with the DNA topoisomerase I inhibitor, camptothecin (CPT), or tumor necrosis factor (TNFα), populations of cells appeared that had the capability to bind FFCK. Most FFCK-binding cells were identified by fluorescence microscopy and laser scanning cytometry (LSC) as the cells undergoing apoptosis. Frequency of cells binding FFCK strongly correlated with frequency of cells having activated caspases (r=0.98 in CPT-treated, and r=0.99 in TNFα-treated cultures). The observed induction of FFCK binding we interpret as representing the activation of a chymotrypsin-like apoptotic Ser protease(s). Pretreatment of cells with the poly-caspase inhibitor, Z-VAD-FMK, prevented the activation of these Ser protease(s). Pretreatment with TPCK, however, had a less pronounced, although distinct and reproducible suppressive effect, on caspase activation. The data, thus, suggest that activation of caspases is an upstream event required for activation of Ser protease(s). Activation of the latter, however, appears to additionally amplify, in a cascade-like mode, caspases activation. Differential color fluorochrome-labeling allowed us to discriminate, within the same cells, between the activation of active caspases and Ser protease(s). Despite a certain degree of co-localization, the inter- and intra-cellular pattern of caspase- vs. Ser-protease(s) was different. Our approach makes it possible to simultaneously monitor activation of caspases and Ser proteases in the same live cells that are induced to apoptosis.