| Monitoring caspase activity in living cells using fluorescent proteins and flow cytometry. | |
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MedLine Citation:
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PMID: 15161627 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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A molecular probe was developed to monitor caspase activity in living cells by flow cytometry. It consists of CFP and YFP with a peptide linker containing two caspase-cleavage sites (LEVD). Its expression resulted in intense fluorescence resonance energy transfer (FRET), whereas cleavage of this linker by caspases eliminated FRET because of physical separation of the CFP and YFP moieties. Using flow cytometry, cells expressing this probe exhibited two patterns, strong FRET and diminished or absent FRET. The appearance of diminished FRET was inhibited by a pan-caspase inhibitor z-VAD or D->A mutations in the LEVD sequence and was markedly increased by apoptosis-inducing agents, etoposide and camptothecin, or overexpression of a caspase 8-red fluorescent protein fusion protein. Importantly, this probe's ability to monitor caspase activity was comparable with results obtained with fluorogenic substrates or fluorochrome-labeled inhibitors of caspases. Specific caspase inhibitors indicated the probe was highly sensitive to cleavage by caspase 6 and 8, less sensitive to caspase 4, and resistant to other caspases. Activation of caspase 8 by Fas engagement markedly increased the probe's cleavage, whereas treatment of caspase 8-deficient cells with anti-Fas did not increase cleavage. However, staurosporine induced cleavage of the probe in caspase 8-deficient cells by a mechanism that was inhibited by overexpression of bcl-x. Taken together, the data indicate that this caspase-sensitive probe can be used to monitor the basal and apoptosis-related activities of caspases, including an initiator caspase, caspase 8, and effector caspases, such as caspase 6. |
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Authors:
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Liusheng He; Xiaoli Wu; Francoise Meylan; Douglas P Olson; James Simone; Derek Hewgill; Richard Siegel; Peter E Lipsky |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: The American journal of pathology Volume: 164 ISSN: 0002-9440 ISO Abbreviation: Am. J. Pathol. Publication Date: 2004 Jun |
Date Detail:
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Created Date: 2004-05-26 Completed Date: 2004-07-08 Revised Date: 2009-11-18 |
Medline Journal Info:
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Nlm Unique ID: 0370502 Medline TA: Am J Pathol Country: United States |
Other Details:
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Languages: eng Pagination: 1901-13 Citation Subset: AIM; IM |
Affiliation:
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Flow Cytometry Section, Office of Science and Technology, National Institute of Arthritis and Musculosketal and Skin Diseases, National Institute of Health, Bethesda, Maryland 20892, USA. Lihe@mail.nih.gov |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Apoptosis
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physiology* Base Sequence Caspases / antagonists & inhibitors, metabolism* Cysteine Proteinase Inhibitors / pharmacology Environmental Monitoring / methods Flow Cytometry / methods Fluorescent Dyes Hela Cells Humans Jurkat Cells Luminescent Proteins / genetics Molecular Sequence Data Oligodeoxyribonucleotides, Antisense Plasmids |
| Chemical | |
Reg. No./Substance:
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0/Cysteine Proteinase Inhibitors; 0/Fluorescent Dyes; 0/Luminescent Proteins; 0/Oligodeoxyribonucleotides, Antisense; EC 3.4.22.-/Caspases |
| Comments/Corrections | |
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