Establishment and Characterization of Ethidium Bromide Resistance in Simian Virus 40-Transformed Hamster Cells

Abstract

This study describes the isolation and subsequent characterization of four mammalian cell lines resistant to ethidium bromide (EB) . Treatment of the simian virus 40-(SV40) transformed hamster cell line F5-1 first led to the establishment of the F2 cell line, which is resistant to 2 ug EB/ml . At this concentration cytochromes c and b are present in almost normal or only slightly diminished amounts, whereas cytochromes a + as show an obvious decrease . The mitochondria of the F2 cell show a normal ultrastructure, not distinct from the parental cell line F5-1, and contain closed circular DNA . The sensitive parental F5-1 cells, however, when exposed to the same dye concentration exhibit the typical EB-induced ultrastructural changes in the mitochondria, and no more component I mitochondrial DNA can be demonstrated . 1 yr after establishment we derived from the F2 cell three more cell lines, resistant against 4, 8, and 16,ug of EB/ml . These cell lines, termed F4, F8, and F16, respectively, also revealed relatively intact-appearing mitochondria, although distinguishable from F5-1 and F2 mitochondria by a more condensed or unorthodox cristae conformation . F4, F8, and F16 cell lines contained closed circular mitochondrial DNA in the same position as that of the parental F5-1 cells, when analyzed in an isopycnic CsCI-EB gradient . A small shoulder at the lower density side of the DNA I peaks was observed . The newly acquired drug resistance of the F cells is hereditarily transmitted to the progeny cells and retained even after a period of growth in EB-free medium .

Spontaneously Immortalized Mouse Embryo Fibroblasts: Growth Behavior of Wild-Type and Vimentin-Deficient Cells in Relation to Mitochondrial Structure and Activity

“…Because the same phenomena, that is, generation of megamitochondria with intraorganellar myelin figures and of condensed mitochondria of irregular shape, were also seen in cultured mammalian cells treated with ethidium bromide (King et al, 1972;Klietmann et al, 1973) or chloramphenicol (Smith et al, 1970;King et al, 1972), potent poisons of mitochondrial transcription and translation, respectively, the changes in mitochondrial morphology noticed in the immortalized vim Ϫ fibroblasts may result from reduced rates of macromolecular synthesis as a direct consequence of ROS-induced mtDNA damage. The varying ultrastructural organization of the mitochondria in the different vim Ϫ cell clones presumably reflects alternative ways of adaptation of mitochondrial structure to metabolic requirements continuously changing during the long phase of senescence under the influence of oxidative stress.…”

Central role of mitochondria in metabolic regulation of liver pathophysiology

“…We found that the cytochrome b 559 is significantly increased by SV40 transformation of cultured cells. Also, in various hepatoma cells such as ascites hepatoma cells and minimal deviation hepatoma cells it increased 13 . It also increases during hepatic carcinogenesis by 3′‐dimethylaminoazo benzene (DAB) in rats.…”

“…Also, in various hepatoma cells such as ascites hepatoma cells and minimal deviation hepatoma cells it increased. 13 It also increases during hepatic carcinogenesis by 3′-dimethylaminoazo benzene (DAB) in rats. They were reported in a series in Cancer Research in 1970-1973.…”

Selection of mammalian cells resistant to a chloramphenicol analog.

“…This technique allowed for the selection of cells which were resistant to chloramphenicol at the level of isolated mitochondrial protein synthesis (8) and whose resistance was a cytoplasmic characteristic (10). Mammalian cells resistant to ethidium bromide have also been reported (11), but the nature of this resistance is not known. Studies of mutants of mitochondrial function are necessary for further elucidation of the nature and function of the mitochondrial genetic system in mammalian cells.…”