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.
Document Type: Research Article
Affiliations: Brander Cancer Research Institute, New York Medical College, Valhalla, NY 10595, USA
Publication date: 01 January 2002
- The International Journal of Oncology provides an international forum for the publication of the latest, cutting-edge research in the broad area of oncology and cancer treatment. The journal accepts original high quality works and reviews on all aspects of oncology research including carcinogenesis, metastasis, epidemiology, chemotherapy and viral oncology. Through fair and efficient peer review, the journal is dedicated to publishing top tier research in the field, offering authors rapid publication as well as high standards of copy-editing and production. The International Journal of Oncology is published on a monthly basis in both print and early online.
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