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

A wide variety of human diseases have been associated with defects in mitoc hondrial DNA (mtDNA). The exact mechanism by which specific mtDNA mutations cause disease is unknown and, although the disparate phenotypes might be e xplained on the basis of impaired mitochondrial gene function alone, the ro le of altered nuclear gene expression must also be considered. In recent ye ars, the experimental technique of depleting cells of mtDNA by culturing th em with ethidium bromide has become a popular method of studying mitochondr ial disorders. However, apart from depleting mtDNA, ethidium bromide may ha ve many other intracellular and nuclear effects. The aim of the present stu dy 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 expr ession between wild-type, mtDNA-replete (rho(+)) and ethidium bromide-treat ed, mtDNA-depleted (rho(0)) cells. Expression of the majority of nuclear-en coded genes, including those for subunits involved in oxidative phosphoryla tion, remained unaffected by the treatment. Seven clones were found to be u nderexpressed; three of the clones showed significant similarity with seque nces of the human genes encoding RNase L inhibitor, human tissue factor and ARCN I (archain vesicle transport protein I), a highly conserved species w hich is related to vesicle structure and trafficking proteins. We conclude that the effects of ethidium bromide treatment on nuclear gene expression a re not simply limited to changes in pathways directly associated with known mitochondrial function. Further studies will be required to elucidate whic h of these changes are due to mtDNA depletion, ATP deficiency or other disp arate effects of ethidium bromide exposure. Given that most genes appear un affected, the results suggest that depleting cells of mtDNA by ethidium bro mide treatment is a valuable approach for the study of mitochondrial mutati ons by cybrid techniques.