Document Detail


Unreliability of the cytochrome c-enhanced green fluorescent fusion protein as a marker of cytochrome c release in cells that overexpress Bcl-2.
MedLine Citation:
PMID:  11489892     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A cytochrome c-enhanced green fluorescent protein chimera (cyt-c.EGFP) was used to monitor the release of cytochrome c from mitochondria in Bcl-2-negative and Bcl-2-positive MDA-MB-468 breast cancer cells. A comparison was made with the intracellular distribution of endogenous cytochrome c based on Western blotting of cell fractions and immunocytochemistry. The release of endogenous cytochrome c from mitochondria into the cytoplasm was detected in Bcl-2-negative cells treated with the kinase inhibitor staurosporine or the calcium-ATPase inhibitor thapsigargin. No release of endogenous cytochrome c was evident in Bcl-2-positive cells, consistent with earlier evidence that Bcl-2 overexpression inhibits cytochrome c release from mitochondria. Cyt-c.EGFP appeared to be localized to the mitochondria in Bcl-2-negative cells and to be released into the cytoplasm following treatment with either staurosporine or thapsigargin. However, in Bcl-2-positive cells the pattern of distribution of cytochrome c-EGFP was inconsistent with that of endogenous cytochrome c, due to accumulation of both cyt-c.EGFP and free EGFP in the cytoplasm of both treated and untreated cells. In summary, cyt-c.EGFP may be useful for monitoring cytochrome c release in living cells that do not express high levels of Bcl-2 but is an unreliable marker of cytochrome c release in cells that overexpress Bcl-2.
Authors:
M Unkila; K S McColl; M J Thomenius; K Heiskanen; C W Distelhorst
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.     Date:  2001-08-06
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  276     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2001 Oct 
Date Detail:
Created Date:  2001-10-15     Completed Date:  2001-12-04     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  39132-7     Citation Subset:  IM    
Affiliation:
Division of Hematology/Oncology, Department of Medicine and Comprehensive Cancer Center at Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106, USA.
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MeSH Terms
Descriptor/Qualifier:
Apoptosis
Blotting, Western
Caspase 3
Caspases / metabolism
Cytochrome c Group / metabolism*
DNA, Complementary / metabolism
Enzyme Activation
Enzyme Inhibitors / pharmacology
Genetic Markers
Green Fluorescent Proteins
Humans
Immunohistochemistry
Luminescent Proteins / metabolism*
Microscopy, Fluorescence
Mitochondria / metabolism
Proto-Oncogene Proteins c-bcl-2 / metabolism*
Recombinant Fusion Proteins / chemistry,  metabolism*
Staurosporine / pharmacology
Subcellular Fractions
Thapsigargin / pharmacology
Time Factors
Transfection
Tumor Cells, Cultured
Grant Support
ID/Acronym/Agency:
CA79806/CA/NCI NIH HHS; CA85804/CA/NCI NIH HHS; T32-CA73515/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Cytochrome c Group; 0/DNA, Complementary; 0/Enzyme Inhibitors; 0/Genetic Markers; 0/Luminescent Proteins; 0/Proto-Oncogene Proteins c-bcl-2; 0/Recombinant Fusion Proteins; 147336-22-9/Green Fluorescent Proteins; 62996-74-1/Staurosporine; 67526-95-8/Thapsigargin; EC 3.4.22.-/CASP3 protein, human; EC 3.4.22.-/Caspase 3; EC 3.4.22.-/Caspases

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