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Labeling of apoptotic JURL-MK1 cells by fluorescent caspase-3 inhibitor FAM-DEVD-fmk occurs mainly at site(s) different from caspase-3 active site
Kuzelová K., Grebenová D., Hrkal Z.
Jazyk angličtina Země Spojené státy americké
NLK
Free Medical Journals
od 2003 do Před 1 rokem
Wiley Online Library (archiv)
od 1980-01-01 do 2012-12-31
Wiley Free Content
od 2003 do Před 1 rokem
- MeSH
- apoptóza MeSH
- barvení a značení MeSH
- chloromethylketony aminokyselin analýza farmakologie metabolismus MeSH
- financování organizované MeSH
- fluorescenční barviva analýza metabolismus MeSH
- inhibitory kaspas MeSH
- kaspasa 3 MeSH
- kinetika MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- podjednotky proteinů metabolismus MeSH
- posttranslační úpravy proteinů účinky léků MeSH
- propidium MeSH
- průtoková cytometrie MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- substrátová specifita MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. Copyright 2007 International Society for Analytical Cytology.
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- $a Labeling of apoptotic JURL-MK1 cells by fluorescent caspase-3 inhibitor FAM-DEVD-fmk occurs mainly at site(s) different from caspase-3 active site / $c Kuzelová K., Grebenová D., Hrkal Z.
- 314 __
- $a Department of Cellular Biochemistry, Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 20 Prague 2, Czech Republic. kuzel@uhkt.cz
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- $a BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. Copyright 2007 International Society for Analytical Cytology.
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