Ethidium bromide as a marker of mtDNA replication in living cells

Abstract

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.

Association of 5-Methylcytosine and 5-Hydroxymethylcytosine with Mitochondrial DNA Content and Clinical and Biochemical Parameters in Hepatocellular Carcinoma

“…Human mitochondrial DNA (mtDNA), a 16.6-kb circular double-stranded DNA molecule that lacks introns and protective histones, is susceptible to oxidative DNA damage and exhibits a high mutation rate [1]. Evidence has suggested that mtDNA may also undergo mutations, insertions or deletions in response to oxidative stress in some cancers [2], [3], and mtDNA in tumour cells was found to play an important role in maintaining the malignant phenotype [4].…”

Hypoxia-induced mitochondrial stress granules

“…EtBr has been shown to label both mitochondrial DNA (nucleoid) and its RNA transcripts in human cancer cells [ 17 – 21 ]. To investigate if the mitoSGs were composed of DNA, RNA, or both, we applied SYBR gold, a DNA-preferring dye [ 22 ], in a co-staining assay with EtBr in worm body wall muscle.…”

“…Although ethidium bromide is most commonly used as a DNA dye in vitro, our data shows that ethidium bromide strongly stains RNA in vivo in C. elegans with much weaker staining of DNA. Ethidium bromide staining of mitochondria has been observed in various cell lines and staining attributed to both DNA and RNA has been reported [ 19 – 21 , 48 ]. In these studies, punctate mitochondrial staining was assumed to be DNA and diffuse staining attributed to mitochondrial RNA although this was rigorously determined for only one cell type [ 19 ].…”

Controle do número de cópias de DNA mitocondrial em células bovinas: um modelo baseado na depleção

“…O EtBr é uma droga amplamente utilizada em modelos de estudo envolvendo o envelhecimento celular e câncer (VILLA et al, 2012). Segundo Nass, 1970, o brometo de etídio gera depleção no número de cópias de mtDNA na célula, devido a uma interferência na incorporação da timidina ao DNA circular.…”

“…Isso ocorre devido à exposição ao EtBr, que afeta a incorporação de timidina ao DNA circular. Outros estudos também mostram que células com baixa contagem de mtDNA tendem a manifestar resistência a quimioterápicos (OHTA, 2006) e que o DNA mitocondrial tem papel importante na manutenção do fenótipo maligno em células tumorais(VILLA et al, 2012).A geração de um modelo animal de depleção do mtDNA em bovinos pode ser utilizado para teste de resistência a quimioterápicos, repleção de mtDNA exógeno por fusão de células depletadas (KHAN; SMIGRODZKI; SWERDLOW, 2007) ou mesmo o desenvolvimento de modelos de mutação de mtDNA de forma semelhante as encontradas em doenças relacionadas ao mtDNA. A grande vantagem do sistema não murinho é que ele tem uma vida mais longa, o que permitiria estudos de efeito do tempo e da senescência a semelhança do que ocorre no humano.As mitocôndrias têm papel importante na homeostase, no metabolismo energético e na morte celular programada(CHIARATTI et al, 2010).…”