The
oxysterol 25-hydroxycholesterol acts both as a regulatory
sterol determining the expression of genes governed by
sterol regulatory elements and as a substrate for 7-alpha-hydroxylase, the first and rate-limiting
enzyme in the
bile acid synthetic pathway. Most wild-type nonhepatic cells are killed by the cytotoxic action of
25-hydroxycholesterol. In contrast, liver cells, which express 7-alpha-hydroxylase activity, are resistant to killing by
25-hydroxycholesterol. We examined the possibility that selection for resistance to
25-hydroxycholesterol might lead to the derivation of a cell line expressing 7-alpha-hydroxylase. A rat
hepatoma cell line (7-alpha-hydroxylase minus) was transfected with human
DNA and screened for resistance to
25-hydroxycholesterol. Although parental
hepatoma cells were all killed within a week, a 25-hydroxycholesterol-resistant cell line (
L35 cells) which showed stable expression of 7-alpha-hydroxylase activity and
mRNA was obtained. These cells exhibited normal inhibition of
cholesterol biosynthesis by
25-hydroxycholesterol. Blocking 7-alpha-hydroxylase activity with
ketoconazole also blocked the resistance of
L35 cells to
25-hydroxycholesterol. Isolation of microsomes from these cells showed levels of 7-alpha-hydroxylase activity (22.9 pmol/min/mg of
protein) that were comparable to the activity (33.2 pmol/min/mg) of microsomes isolated from the livers of rats killed during the high point of the diurnal cycle. Parental cells had no detectable activity. These data show a new complementation group for
25-hydroxycholesterol resistance: expression of 7-alpha-hydroxylase.
Dexamethasone increased both the activity and the cellular content of
mRNA coding for 7-alpha-hydroxylase. Since
dactinomycin blocked the ability of
dexamethasone to induce
mRNA, active transcription is required. Southern analysis of genomic
DNA showed that
L35 cells contain the rat (endogenous) gene but not the human gene. Furthermore, the
RNA expressed by
L35 cells is similar in size to rat
RNA and is distinct from the human form of 7-alpha-hydroxylase. The combined data indicate that
L35 cells are resistant to
25-hydroxycholesterol because they express 7-alpha-hydroxylase. The mechanism responsible involves activation of the endogenous (silent) gene of the parental rat
hepatoma cell.