title: Ethidium bromide as a marker of mtDNA replication in living cells.
abstract: 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.affiliation: University of Milano-Bicocca, Department of Biotechnology e Biosciences, P.za della Scienza 2, 20126 Milan, Italy. annamaria.villa@unimib.it
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Villa
authors.forename: Paola
authors.initials: P
authors.lastname: Fusi
authors.forename: Valentina
authors.initials: V
authors.lastname: Pastori
authors.forename: Giulia
authors.initials: G
authors.lastname: Amicarelli
authors.forename: Chiara
authors.initials: C
authors.lastname: Pozzi
authors.forename: Daniel
authors.initials: D
authors.lastname: Adlerstein
authors.forename: Silvia Maria
authors.initials: SM
authors.lastname: Doglia
mesh_terms: D001973:Bromodeoxyuridine / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000097:blood; D004996:Ethidium / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; D005456:Fluorescent Dyes; D004996:Ethidium; D001973:Bromodeoxyuridine