Differential expression of mRNA in human thyroid cells depleted of mitochondrial DNA by ethidium bromide treatment

Abstract

A wide variety of human diseases have been associated with defects in mitochondrial DNA (mtDNA). The exact mechanism by which specific mtDNA mutations cause disease is unknown and, although the disparate phenotypes might be explained on the basis of impaired mitochondrial gene function alone, the role of altered nuclear gene expression must also be considered. In recent years, the experimental technique of depleting cells of mtDNA by culturing them with ethidium bromide has become a popular method of studying mitochondrial disorders. However, apart from depleting mtDNA, ethidium bromide may have many other intracellular and nuclear effects. The aim of the present study was to investigate the effects of ethidium bromide treatment on nuclear gene expression. A simian-virus-40-transformed human thyroid cell line was depleted of mtDNA by culture in ethidium bromide, and differential display reverse transcriptase-PCR (DDRT-PCR) was then employed to compare mRNA expression between wild-type, mtDNA-replete (rho(+)) and ethidium bromide-treated, mtDNA-depleted (rho(0)) cells. Expression of the majority of nuclear-encoded genes, including those for subunits involved in oxidative phosphorylation, remained unaffected by the treatment. Seven clones were found to be underexpressed; three of the clones showed significant similarity with sequences of the human genes encoding RNase L inhibitor, human tissue factor and ARCN1 (archain vesicle transport protein 1), a highly conserved species which is related to vesicle structure and trafficking proteins. We conclude that the effects of ethidium bromide treatment on nuclear gene expression are not simply limited to changes in pathways directly associated with known mitochondrial function. Further studies will be required to elucidate which of these changes are due to mtDNA depletion, ATP deficiency or other disparate effects of ethidium bromide exposure. Given that most genes appear unaffected, the results suggest that depleting cells of mtDNA by ethidium bromide treatment is a valuable approach for the study of mitochondrial mutations by cybrid techniques.