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Ethidium bromide as a marker of mtDNA replication in living cells.
Villa, Anna Maria; Fusi, Paola; Pastori, Valentina; Amicarelli, Giulia; Pozzi, Chiara; Adlerstein, Daniel; Doglia, Silvia Maria.
Afiliação
  • Villa AM; University of Milano-Bicocca, Department of Biotechnology e Biosciences, P.za della Scienza 2, 20126 Milan, Italy. annamaria.villa@unimib.it
J Biomed Opt ; 17(4): 046001, 2012 Apr.
Article em En | MEDLINE | ID: mdl-22559680
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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: DNA Mitocondrial / Replicação do DNA / Etídio / Corantes Fluorescentes Tipo de estudo: Prognostic_studies Limite: Humans Idioma: En Revista: J Biomed Opt Assunto da revista: ENGENHARIA BIOMEDICA / OFTALMOLOGIA Ano de publicação: 2012 Tipo de documento: Article País de afiliação: Itália País de publicação: EEUU / ESTADOS UNIDOS / ESTADOS UNIDOS DA AMERICA / EUA / UNITED STATES / UNITED STATES OF AMERICA / US / USA

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: DNA Mitocondrial / Replicação do DNA / Etídio / Corantes Fluorescentes Tipo de estudo: Prognostic_studies Limite: Humans Idioma: En Revista: J Biomed Opt Assunto da revista: ENGENHARIA BIOMEDICA / OFTALMOLOGIA Ano de publicação: 2012 Tipo de documento: Article País de afiliação: Itália País de publicação: EEUU / ESTADOS UNIDOS / ESTADOS UNIDOS DA AMERICA / EUA / UNITED STATES / UNITED STATES OF AMERICA / US / USA