Isolation and characterization of Chinese hamster ovary cells defective in the intracellular metabolism of low density lipoprotein-derived cholesterol.
We have isolated clones of an established cell line which express defects in intracellular cholesterol metabolism. Chinese hamster ovary cells were mutagenized, and clones unable to mobilize low density lipoprotein (LDL)-derived cholesterol to the plasma membrane were selected. Biochemical analysis of two mutant clones revealed a phenotype characteristic of the lysosomal storage disease, Niemann-Pick type C. The mutant cell lines were found to be defective in the regulatory responses elicited by LDL-derived cholesterol. LDL-mediated stimulation of cholesterol esterification was grossly defective, and LDL suppression of 3-hydroxy-3-methylglutaryl-CoA reductase was impaired. However, the mutants modulated these activities normally in response to 25-hydroxycholesterol or mevalonate. The LDL-specific defects were predicated by the inability of these mutants to mobilize LDL-derived cholesterol from lysosomes. Cell fractionation studies showed that LDL-derived, unesterified cholesterol accumulated in the lysosomes of mutant cells to significantly higher levels than normal, commensurate with defective movement of cholesterol to other cellular membranes. Characterization of cell lines defective in intracellular cholesterol transport will facilitate identification of the gene(s) required for intracellular cholesterol movement and regulation.
Dahl
N K
NK
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Reed
K L
KL
Daunais
M A
MA
Faust
J R
JR
Liscum
L
L
D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D008078:Cholesterol, LDL / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus / Q000235:genetics; D006888:Hydroxycholesterols / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008247:Lysosomes / Q000378:metabolism; D008798:Mevalonic Acid / Q000494:pharmacology
D008078:Cholesterol, LDL; D006888:Hydroxycholesterols; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; C007997:25-hydroxycholesterol; D000666:Amphotericin B; D006903:Hydroxymethylglutaryl CoA Reductases; D008798:Mevalonic Acid