From: DARZYNKIEWICZ ZBIGNIEW (DARZYNK@nymc.edu)
Date: Sun Mar 02 2003 - 12:44:02 EST
Dear Colleagues, During the past two years we and other authors were publishing on application of the fluorochrome-labeled inhibitors of caspases (e.g. FAM-VAD-FMK, FITC-VAD-FMK), and more recently of serine proteases (FFCK, FLCK), in studies of apoptosis. These reagents are available from several vendors under the trade names such as CaspGLOW, CaspaTag, FLICA and FLISP. Binding of these inhibitors, which were designed as affinity labels of the enzyme active centers, has been interpreted as reflecting activation of caspases and serine proteases during apoptosis, respectively. These reagents are convenient, specific and sensitive markers of apoptotic cells and in combination with e.g. the exclusion of propidium iodide assay as a probe of plasma membrane integrity, they reveal three sequential stages of apoptosis. Very recently, however, additional control experiments have been done and we observed in some cell types that a significant component in binding of these reagents to apoptotic cells may not reflect their direct reactivity with the protease's active centers. In collaboration with Brian Lee and Gary Johnson from Immunochemistry Technologies experiments are being done to further explain degree of specificity and other modes of binding of these reagents to apoptotic cells (to be published). However, to alert these researchers who may be using these inhibitors we are prompted to use the Cytometry Internet Board to reveal as soon as it is possible the problems related to interpretation of the data obtained with the use of these reagents. While these reagents are good markers of apoptosis and very likely report activation of caspases or serine proteases, we currently suspect that mechanism of their retention in apoptotic cells may not be a strict function of their covalent interaction with caspase or protease enzymes and may, in fact, involve other mechanisms as well. Zbigniew Darzynkiewicz Brian Lee Gary Johnson
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