Isolation and preliminary characterization of rat liver cells resistant to 7-ketocholesterol

Two clones of the rat liver cell line, GAI, were selected and propagated in 4 microgram/ml of the cholesterol analogue, 7-ketocholesterol. One of the variants was also found to be resistant to another toxic analogue, 25-hydroxycholesterol. 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) activity was elevated in both variant clones, but was not increased substantially upon incubation of the cells in lipid-depleted medium. The reductase activity was increased 3-fold in the wild type cells under the same conditions. Incubation of the cells in lipid-depleted medium resulted in a 3-fold increase in the rate of sterol synthesis from [14C]acetate in all three types. In the presence of 2 microgram/ml 7-ketocholesterol, the reductase activity in all three cell lines decreased at an equal rate, but activity of the enzyme was still measurable after 48 h in the presence of the analogue. Sterol synthesis was reduced almost to zero in the wild type cells under these conditions, while the variant cells retained 12--15% of their capacity for sterol synthesis. These results are interpreted to indicate that 7-ketocholesterol, besides affecting sterol synthesis at the level to 3-hydroxy-3-methylglutaryl coenzyme A reductase, must also affect sterol synthesis at another step further along the synthetic pathway.