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Ethidium bromide as a marker of mtDNA replication in living cells.
MedLine Citation:
PMID:  22559680     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity. To prove this hypothesis, in the present work we studied, by LSCFM, the EB fluorescence in mitochondria nucleoids of living neuroblastoma cells, a model system in which differentiation affects the level of mtDNA replication. A drastic decrease of fluorescence was observed after differentiation. To correlate EB fluorescence intensity to the mtDNA replication state, we evaluated the mtDNA nascent strands content by ligation-mediated real-time PCR, and we found a halved amount of replicating mtDNA molecules in differentiating cells. A similar result was obtained by BrdU incorporation. These results indicate that the low EB fluorescence of nucleoids in differentiated cells is correlated to a low content of replicating mtDNA, suggesting that EB may be used as a marker of mtDNA replication in living cells.
Authors:
Anna Maria Villa; Paola Fusi; Valentina Pastori; Giulia Amicarelli; Chiara Pozzi; Daniel Adlerstein; Silvia Maria Doglia
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of biomedical optics     Volume:  17     ISSN:  1560-2281     ISO Abbreviation:  J Biomed Opt     Publication Date:  2012 Apr 
Date Detail:
Created Date:  2012-05-07     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9605853     Medline TA:  J Biomed Opt     Country:  United States    
Other Details:
Languages:  eng     Pagination:  046001     Citation Subset:  IM    
Affiliation:
University of Milano-Bicocca, Department of Biotechnology e Biosciences, P.za della Scienza 2, 20126 Milan, Italy.
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