Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. I. Characterization and the respiratory enzymes.

Cell lines resistant to ethidium bromide have been developed from cultured mammalian BHK21/C13 cells and these same cells transformed by Rous sarcoma virus (C13/B4). Cells resistant to 2 micrograms ethidium bromide per milliliter have been cloned. One clone of the control and one of the virus-transformed cell lines has been employed for characterization. The resistant cells, in the presence of 2 micrograms ethidium bromide/ml, grow at approximately the same rate as the untreated parental cells. The control cells possess a "normal" karyotype (44 chromosomes), while the corresponding ethidium bromide mutant has a reduced chromosome number of 41 and a number of translocations. The mitochondria displayed morphological alterations compared to the parental lines during the transition phase prior to the isolation of the ethidium bromide-resistant cells. The mitochondria of the ethidium bromide-resistant mutants appear somewhat enlarged with a normal morphology. The effect of ethidium bromide on selected respiratory enzymes in normal and virus-transformed ethidium bromide-resistant baby hamster kidney cells was determined. Ethidium bromide-resistant cells exhibited a depressed level of cytochrome aa3. This depression could not be reversed by growth in ethidium bromide-free media. Ethidium bromide-resistant cells possessed the same cytochrome b, c, and c1 levels per cell as their corresponding parental lines. Purified mitochondria isolated from virus-transformed ethidium bromide-resistant cells exhibited a depression in cytochrome oxidase-specific activity, while the ethidium bromide-resistant control cells did not. All cell lines studied showed a depression in NADH-ferricyanide and NADH-cytochrome c reductase-specific activities relative to their parental BHK21/C13 cells. No increase was observed in virus-transformed ethidium bromide-resistant cells. Ethidium bromide-resistant control cells exhibited a two-fold increase in oligomycin-insensitive adenosine triphosphatase activity relative to their parental cells. All of the cell lines studied possessed equivalent oligomycin-sensitive adenosine triphosphatase-specific activity except for the virus-transformed, dye-resistant mutant, whose activity was increased.