Document Detail


Monitoring caspase activity in living cells using fluorescent proteins and flow cytometry.
MedLine Citation:
PMID:  15161627     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Liusheng He; Xiaoli Wu; Francoise Meylan; Douglas P Olson; James Simone; Derek Hewgill; Richard Siegel; Peter E Lipsky
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  The American journal of pathology     Volume:  164     ISSN:  0002-9440     ISO Abbreviation:  Am. J. Pathol.     Publication Date:  2004 Jun 
Date Detail:
Created Date:  2004-05-26     Completed Date:  2004-07-08     Revised Date:  2013-06-09    
Medline Journal Info:
Nlm Unique ID:  0370502     Medline TA:  Am J Pathol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1901-13     Citation Subset:  AIM; IM    
Affiliation:
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
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MeSH Terms
Descriptor/Qualifier:
Apoptosis / physiology*
Base Sequence
Caspase Inhibitors
Caspases / 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:
0/Caspase Inhibitors; 0/Cysteine Proteinase Inhibitors; 0/Fluorescent Dyes; 0/Luminescent Proteins; 0/Oligodeoxyribonucleotides, Antisense; EC 3.4.22.-/Caspases
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