Characterization of a Chinese Hamster Ovary Cell Line Resistant to Uncouplers

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Abstract
The chemiosmotic theory of oxidative phosphorylation and the action of uncouplers was examined by characterizing a clone, UH5, of Chinese hamster ovary (CHO TK−) cells resistant to 5‐chloro‐3‐tert‐butyl‐2′‐chloro‐4′‐nitrosalicylanilide (S‐13), a potent uncoupler of oxidative phosphorylation. About 9‐times and 4‐times more S‐13 was required to effect growth and respiration respectively of UH5 cells compared to the parental CHO TK− cells. UH5 cells were cross‐resistant to the uncouplers SF‐6847 (3,5‐di‐tert‐butyl‐4‐hydroxy‐benzylidenemalononitrile), carbonylcyanide p‐trifluoromethoxyphenylhydrazone and 2,4‐dinitrophenol but not to oligomycin, venturicidin or Tevenel. Size, chromosome number and DNA content indicated that the UH5 cell line was probably pseudotetraploid compared to the parental pseudodiploid CHO TK− cells. Hybrid and cybrid cells formed from crosses of UH5 cells and cytoplasts, respectively, with an uncoupler‐sensitive cell line were sensitive to S‐13 indicating that resistance is probably nuclear‐determined. UH5 cell mitochondria had increased cytochrome oxidase and decreased H+‐ATPase activities. A fivefold resistance of oxidative phosphorylation to uncouplers was found at the mitochondrial level with respiration driven by either succinate or ascorbate/N,N,N′,N′‐tertramethyl‐p‐phenylenediamine. In contrast, no difference in sensitivity was found to valinomycin between mitochondria from UH5 and CHO TK− cells. The oligomycin‐sensitive H+‐ATPase activity of UH5 and CHO TK− cell mitochondria was equally stimulated by the uncoupler S‐13. Uncoupler‐resistant mitochondria would not be expected on the basis of the chemiosmotic theory, and the relation of the results to other modes of coupling is considered.

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