Activation of the silent endogenous cholesterol-7-alpha-hydroxylase gene in rat hepatoma cells: a new complementation group having resistance to 25-hydroxycholesterol.
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.
Leighton
J K
JK
Atherosclerosis and Hepatobiliary Research Center, University of Colorado Health Sciences Center, Denver 80262.
Dueland
S
S
Straka
M S
MS
Trawick
J
J
Davis
R A
RA
D000818:Animals; D002784:Cholesterol / Q000096:biosynthesis; D002790:Cholesterol 7-alpha-Hydroxylase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D005786:Gene Expression Regulation; D005796:Genes; D005816:Genetic Complementation Test; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007654:Ketoconazole / Q000494:pharmacology; D008077:Lipoproteins, LDL / Q000494:pharmacology; D008114:Liver Neoplasms, Experimental; D008862:Microsomes, Liver / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D006888:Hydroxycholesterols; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; C007997:25-hydroxycholesterol; D003907:Dexamethasone; D002784:Cholesterol; D002790:Cholesterol 7-alpha-Hydroxylase; D007654:Ketoconazole