Kuzelová Katerina, Grebenová Dana, Hrkal Zbynek
Department of Cellular Biochemistry, Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 20 Prague 2, Czech Republic.
Cytometry A. 2007 Aug;71(8):605-11. doi: 10.1002/cyto.a.20415.
Fluorochrome-labeled inhibitors of caspases (FLICA) have been designed as an alternative tool for the detection of caspase activation in whole cells. They should label the active site of the corresponding caspase through a covalent attachment to the reactive cysteine residue.
One of the FLICAs, FAM-DEVD-fmk, was used to monitor apoptosis progression in leukemic JURL-MK1 cells by means of flow cytometry. The effects of unlabeled caspase inhibitors z-DEVD-fmk and z-VAD-fmk on FLICA staining were analyzed to evaluate the contribution of caspase-bound FLICA to the fluorescent signal. Covalent binding of inhibitors to caspase-3 subunit was revealed by Western blotting.
Although the unlabeled inhibitors irreversibly bind to caspase-3, completely inhibit its activity, and prevent FLICA binding to caspase-3 even at concentrations lower than 5 muM, they have no effect on FLICA staining of apoptotic cells.
Fluorescent signal of FLICA is characteristic for apoptotic cells but originates mainly from yet unspecified site(s) that differ from the caspase active site. This finding puts in doubt the specificity of staining by various FLICAs with regard to individual caspases and shows the need for an extreme care in the interpretation of data obtained using these labels.
荧光素标记的半胱天冬酶抑制剂（FLICA）已被设计为检测全细胞中半胱天冬酶激活的替代工具。它们应通过与反应性半胱氨酸残基的共价连接来标记相应半胱天冬酶的活性位点。
使用一种FLICA（FAM-DEVD-fmk）通过流式细胞术监测白血病JURL-MK1细胞中的凋亡进程。分析未标记的半胱天冬酶抑制剂z-DEVD-fmk和z-VAD-fmk对FLICA染色的影响，以评估与半胱天冬酶结合的FLICA对荧光信号的贡献。通过蛋白质印迹法揭示抑制剂与半胱天冬酶-3亚基的共价结合。
尽管未标记的抑制剂与半胱天冬酶-3不可逆结合，完全抑制其活性，甚至在浓度低于5μM时就能阻止FLICA与半胱天冬酶-3结合，但它们对凋亡细胞的FLICA染色没有影响。
FLICA的荧光信号是凋亡细胞的特征，但主要来自与半胱天冬酶活性位点不同的尚未明确的位点。这一发现质疑了各种FLICA针对单个半胱天冬酶染色的特异性，并表明在解释使用这些标记获得的数据时需要格外谨慎。
