Isolation of Chinese hamster ovary cell lines expressing human acyl-coenzyme A/cholesterol acyltransferase activity.

Abstract

Abstract. We have previously reported the isolation of Chinese hamster ovary cell mutants deficient in acylcoenzyme A/cholesterol acyltransferase (ACAT) activity (Cadigan, K. M., J. G. Heider, and T. Y. Chang. 1988. J. Biol. Chem. 263:274-282). We now describe a procedure for isolating cells from these mutants that have regained the ability to synthesize cholesterol esters. The protocol uses the fluorescent stain Nile red, which is specific for neutral lipids such as cholesterol ester. After ACAT mutant populations were subjected to chemical mutagenesis or transfected with human fibroblast whole genomic DNA, two revertants and one primary transformant were isolated by virtue of their higher fluorescent intensities using flow cytofluorimetry. Both the revertants and transformant have regained large amounts of intracellular cholesterol ester and ACAT activity. However, heat inactivation experiments revealed that the enzyme activity of the transformant had heat stability properties identical to that of human fibroblasts, while the ACAT activities of the revertants were similar to that of other Chinese hamster ovary cell lines. These results suggest that the molecular lesion in the ACAT mutants resides in the structural gene for the enzyme, and the transformant has corrected this defect by acquiring and stably expressing a human gene encoding the ACAT polypeptide. Secondary transformants were isolated by transfection of ACAT mutant cells with primary transformant genomic DNA. Genomic Southern analysis of the secondary transformants using a probe specific for human DNA revealed several distinct restriction fragments common to all the transformants which most likely comprise part or all of the human ACAT gene. The cell lines described here should facilitate the cloning of the gene encoding the human ACAT enzyme.vL-coenzyme A/cholesterol acyltransferase (ACAT) ~ is an intracellular enzyme that uses cholesterol and fatty acyl-coenzyme A (CoA) to form cholesterol esters (10, 50). The enzyme is localized to the rough endoplasmic reticulum in rat liver (2, 24); is highly regulated in many cell types and tissues; and is believed to play an important role in cholesterol metabolism in various cells and tissues such as the small intestinal mucosa, hepatocytes, and the steroid hormone-producing tissues (10, 50).Although ACAT has been studied intensively, little is known about its molecular structure. In rat liver, the active site of the enzyme has been localized to the cytoplasmic surface of the microsomal vesicles using a combination of detergent and protease treatments (24, 34), but whether the enzyme spans the entire membrane could not be determined. Recent chemical modification studies have demonstrated that an essential histidyl and sulfhydryl residue(s) may reside at or near the active site of the enzyme. ACAT activities from different rabbit tissues have different sensitivities to the histidyl-modifying reagents, suggesting the existence of different ACAT subtypes (31, 32). ACAT activity has been solubilized and reconst...

Intracellular cholesterol mobilization involved in the ABCA1/apolipoprotein-mediated assembly of high density lipoprotein in fibroblasts

“…4C), consistent with our previous finding in mouse peritoneal macrophages using a different ACAT inhibitor (35). To avoid the use of ACAT inhibitors that may cause nonspecific side effect(s), we examined ACAT-1-deficient cells, AC29 (25), and AC29 stably expressing human ACAT-1 (AC29/ hACAT1) were also examined to assess the role of ACAT. Table 1 shows cellular cholesterol and ACAT activity in these CHO mutants.…”

“…Human embryonic kidney-derived cell line HEK293 and a clone of its stable human ABCA1-green fluorescent protein transfectant (293/2c) were maintained in DMEM with 10% FBS as reported (22,23), and this clone has been extensively studied (22)(23)(24). ACAT-1-deficient CHO cells, AC29 (25), its parental 25RA cells (26), and AC29 stably expressing human ACAT-1 (AC29/hACAT1) were grown in a 1:1 mixture of DMEM and Ham's F12 supplemented with 10% FBS plus 10 g/ml gentamycin. 25RA cells have a gain-offunction mutation in SREBP cleavage-activating protein, resulting in constitutive activation of the proteolytic cleavage of SREBPs (27).…”

Acyl-coenzyme A:cholesterol acyltransferase promotes oxidized LDL/oxysterol-induced apoptosis in macrophages

“…Previous reports from two laboratories have described the isolation of ACAT defective CHO-K1 mutants with selection for resistance to killing by 25-OHC (54,55). One of these studies also described partial resistance to 25-OHC cytotoxicity in wild-type CHO-K1 cells treated with 58-035 (54).…”

Absence of Nceh1 augments 25-hydroxycholesterol-induced ER stress and apoptosis in macrophages

“…This property has been utilized to isolate mutant cells which are defective in the molecular pathway mediating the sterol-mediated feedback inhibition of cholesterol biosynthesis ( 45 ). Subsequent works have identifi ed molecular defects of these mutant CHO cells, not only in SREBP-2 ( 46, 47 ) and SREBP cleavageactivating protein ( 48 ), but also in ACAT ( 49,50 ). In addition to defective feedback repression of cholesterol biosynthesis, absence of ACAT may be advantageous to antagonize certain toxic effects of 25-HC through a pathway distinct from 25-HC-mediated suppression of cholesterol biosynthesis.…”