Summary
Cells of the human line VA2-B in suspension culture have been treated with very low concentrations of ethidium bromide for the purpose of reducing the amount of mitochondrial DNA (mit-DNA) per cell. Cells maintained in the presence of 5 ng/ml ethidium bromide grew at a normal rate for three days; thereafter, their doubling time gradually increased to a stable value of about 60 h. In these cells, the rate of 3H thymidine incorporation into mit-DNA decreased very rapidly to ∼60% of the normal, and remained thereafter at this level, while the amount of mit-DNA per cell stabilized around a level of 70–80% of the control. In cells long-term treated with 5 ng/ml ethidium bromide, the rate of mitochondrial protein synthesis was about 35% of the normal, and the cytochrome c oxidase activity about 50% of the control. Cells treated with 20 ng/ml of the drug underwent 3–4 cell doublings at control rates, then gradually stopped growing, and eventually died. In these cells, the rate of incorporation of 3H thymidine into mit-DNA was reduced to 50% of the control value after 10 min treatment with ethidium bromide, and became barely detectable after three cell doublings. At this time, the cells had on the average less than 10% of the control amount of mit-DNA, the rate of mitochondrial protein synthesis was reduced to 3% of the normal, and the specific activities of cytochrome c oxidase and rutamycin-sensitive ATPase were less than 20% of the control values. In spite of these marked changes, the cells exhibited only a 20–30% loss in cell viability, as estimated by cloning efficiency, after three days of exposure to the drug. Cells treated with ethidium bromide at 20 ng/ml for three days, and then transferred to drug-free medium, recovered a near-to-normal growth rate and cloning efficiency and a near-to-normal rate of synthesis and amount of mit-DNA in about five days.
Similar content being viewed by others
References
Attardi, B., Cravioto, B., Attardi, G.: Membrane-bound ribosomes in HeLa cells. The proportion to total cell ribosomes and their association with messenger RNA. J. molec. Biol. 44, 47–70 (1969)
Attardi, G., Costantino, P., England, J.M., Lynch, D., Ojala, D., Posakony, J., Storrie, B.: Mitochondrial and nuclear gene interation in HeLa cells. In: Genetic Interaction and Gene Transfer. Brookhaven Symposium in Biology, No. 29, pp. 16–35 (1977)
Berezney, R., Macaulay, L.K., Crane, F.L.: The purification and biochemical characterization of bovine liver nuclear membranes. J. biol. Chem. 247, 5549–5561 (1972)
Berk, A.J., Clayton, D.A.: Mechanism of mitochondrial DNA replication in mouse L-cells: asynchronous replication of strands, segregation of circular daughter molecules, aspects of topology and turnover of an initiation sequence. J. molec. Biol. 86, 801–824 (1974)
Bogenhagen, D., Clayton, D.A.: The number of mitochondrial deoxyribonucleic acid genomes in mouse L and human HeLa cells. Quantitative isolation of mitochondrial deoxyribonucleic acid. J. biol. Chem. 249, 7991–7995 (1974)
Bunn, C.L., Wallace, D.C., Eisenstadt, J.M.: Cytoplasmic inheritance of chloramphenicol resistance in mouse tissue culture cells. Proc. nat. Acad. Sci. (Wash.) 71, 1681–1685 (1974)
Carré, D. and Attardi, G.: Biochemical and electron microscopic characterization of DNA-RNA complexes from HeLa cell mitochondria. Biochemistry 17, 3263–3273 (1978)
Costantino, P., Attardi, G.: Identification of discrete electrophoretic components among the products of mitochondrial protein synthesis in HeLa cells. J. molec. Biol. 96, 291–306 (1975)
Dulbecco, R., Freeman, G.: Plaque production by the polyoma virus. Virology 8, 396–397 (1959)
Englard, S., Siegel, L.: Mitochondrial L-malate dehydrogenase from beef heart. In: Methods in enzymology, 13(S.P. Colwick and N.O. Kaplan, eds.), pp. 99–106. New York: Academic Press 1969
Faye, G., Fukuhara, H., Grandchamp, G., Lazowska, J., Rabinowitz, M., Bolotin-Fukuhara, M., Coen, P., Deutsch, D., Dujon, B., Netter, P., Slonimski, P.: Mitochondrial nucleic acids in the petite colony mutants: deletions and repetitions of genes. Biochimie 55, 779–792 (1973)
Foury, F., Tzagoloff, A.: Localization of mitochondrial DNA of mutations leading to a loss of rutamycin-sensitive adenosine triphosphatase. Europ. J. Biochem. 68, 113–119 (1976)
Koch, G.: Synthesis of the mitochondrial inner membrane in cultured Xenopus laevis oocytes. J. biol. Chem. 251, 6097–6107 (1976)
Kroon, A.M.: Protein synthesis in mitochondria. 3. On the effects of inhibitors on the incorporation of amino acids into protein by intact mitochondria and digitonin fractions. Biochim. biophys. Acta (Amst.) 108, 275–284 (1965)
Lederman, M., Attardi, G.: In vitro protein synthesis in a mitochondrial fraction from HeLa cells: sensitivity ot antibiotics and ethidium bromide. Biochem. biophys. Res. Commun. 40, 1492–1500 (1970)
Leibowitz, R.D.: The effect of ethidium bromide on mitochondrial DNA synthesis and mitochondrial DNA structure in HeLa cells. J. Cell Biol. 51, 116–122 (1971)
Levintow, L., Darnell, J.E.: A simplified procedure for purification of large amounts of polio virus: characterization and amino acid analysis of type I polio virus. J. biol. Chem. 235, 70–73 (1960)
Linnane, A.M., Haslam, J.M.: The biogenesis of yeast mitochondria. In: Current topics in cellular regulation, 2 (B. Horecker and E. Stadtman, eds.), pp. 101–171. New York: Academic Press 1970
Linnane, A.M., Lamb, A.J., Christodoulou, C.: The biogenesis of mitochondria. VI. Biochemical basis of the resistance of Saccharomyces cerevisiae toward antibiotics which specifically inhibit mitochondrial protein synthesis. Proc. nat. Acad. Sci. (Wash.) 59, 1288–1293 (1968)
Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)
Martin, J.B., Doty, D.M.: Determination of inorganic phosphate. Anal. Chem. 21, 965–967 (1949)
Mitchell, C.M., England, J.M., Attardi, G.: Isolation of chloramphenicol-resistant variants from a human cell line. Som. Cell Genet. 1, 215–234 (1975)
Munkres, K.D., Richards, F.M.: Genetic alteration of Neurospora malate dehydrogenase. Arch. Biochem. Biophys. 109, 457–465 (1965)
Nass, M.M.: Differential effects of ethidium bromide on mitochondrial and nuclear DNA synthesis in vivo in cultured mammalian cells. Exp. Cell Res. 72, 211–222 (1972)
Naum, Y., Pious, D.A.: Reversible inhibition of cytochrome oxidase accumulation in human cells by ethidium bromide. Exp. Cell Res. 65, 355–359 (1971)
Ojala, D., Attardi, G.: Expression of the mitochondrial genome in HeLa cells. X. Properties of mitochondrial polysomes. J. molec. Biol. 65, 273–289 (1972)
Perlman, S., Penman, S.: Mitochondrial protein synthesis: resistance to emetine and response to RNA synthesis inhibitors. Biochem. biophys. Res. Commun. 40, 941–948 (1970)
Pontén, J., Jensen, F., Koprowski, H.: Morphological and virological investigation of human tissue cultures transformed with SV40. J. cell comp. Physiol. 61, 145–163 (1963)
Radsak, K., Kato, K., Sato, N., Koprowski, H.: Effect of ethidium bromide on mitochondrial DNA and cytochrome synthesis in HeLa cells. Exp. Cell Res. 66, 410–416 (1971)
Schatz, G., Mason, T.: The biosynthesis of mitochondrial proteins. In: Annual reviews of biochemistry, 43 (E.E. Snell, ed.), pp. 51–88. Palo Alto, California: Annual Reviews, Inc. 1974
Sebald, W., Machleidt, W., Otto, J.: Products of mitochondrial protein synthesis in Neurospora crassa. Determination of equimolar amounts of three products in cytochrome oxidase on the basis of amino acid analysis. Europ. J. Biochem. 38, 311–324 (1973)
Slonimski, P.P., Tzagoloff, A.: Localization in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme QH2-cytochrome c reductase. Europ. J. Biochem. 61, 27–41 (1976)
Smith, C.A., Jordan, J.M., Vinograd, J.: In vivo effects of intercalating drugs on the superhelix density of mitochondrial DNA isolated from human and mouse cells in culture. J. molec. Biol. 59, 255–272 (1971)
Storrie, B., Attardi, G.: Expression of the mitochondrial genome in HeLa cells. XIII. Effect of selective inhibition of cytoplasmic or mitochondrial protein synthesis on mitochondrial nucleic acid synthesis. J. molec. Biol. 71, 177–199 (1972)
Storrie, B., Attardi, G.: Expression of the mitochondrial genome in HeLa cells. IX. Effect of inhibition of mitochondrial protein synthesis on mitochondrial formation. J. Cell Biol. 56, 819–831 (1973)
Tzagoloff, A., Foury, F., Akai, A.: Assembly of the mitochondrial membrane system. XVIII. Genetic loci on mitochondrial DNA involved in cytochrome b biosynthesis. Molec. gen. Genet. 149, 33–42 (1976)
Wallace, D.C., Bunn, C.L., Eisenstadt, J.M.: Cytoplasmic transfer of chloramphenicol resistance in human tissue culture cells. J. Cell Biol. 67, 174–188 (1975)
Wurtz, E.A., Boynton, J.E., Gillham, N.W.: Perturbation of chloroplast DNA amounts and chloroplast gene transmission in Chlamydomonas reinhardtii by 5-fluorodeoxyuridine. Proc. nat. Acad. Sci. (Wash.) 74, 4552–4556 (1977)
Zylber, E., Vesco, C., Penman, S.: Selective inhibition of the synthesis of mitochondria-associated RNA by ethidium bromide. J. molec. Biol. 44, 195–204 (1969)
Author information
Authors and Affiliations
Additional information
Communicated by E. Bautz
Rights and permissions
About this article
Cite this article
Wiseman, A., Attardi, G. Reversible tenfod reduction in mitochondrial DNA content of human cells treated with ethidium bromide. Molec. Gen. Genet. 167, 51–63 (1978). https://doi.org/10.1007/BF00270321
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00270321