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J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.M500198-JLR200 on July 1, 2005

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Journal of Lipid Research, Vol. 46, 1840-1848, September 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Silencing of the mutant SCAP allele accounts for restoration of a normal phenotype in CT60 cells selected for NPC1 expression

Jean Ann Maguire and Jerry W. Reagan, Jr.1

Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC 27157

Published, JLR Papers in Press, July 1, 2005. DOI 10.1194/jlr.M500198-JLR200

1 To whom correspondence should be addressed. e-mail: jreagan{at}mtsu.edu

The sterol regulatory element binding protein (SREBP)/SREBP cleavage-activating protein (SCAP) complex regulates the transcription of numerous genes involved in cellular cholesterol metabolism. The CHO mutant, CT60, and its parental cell line, 25RA, possess a gain-of-function mutation in one allele of the SCAP gene that renders the cells resistant to sterol-mediated suppression of cholesterol synthesis and uptake. In addition, CT60 cells do not express a functional Niemann-Pick type C1 (NPC1) protein, which leads to lysosomal accumulation of free cholesterol. Correction of the NPC1 defect by expression of a yeast artificial chromosome (YAC) containing the NPC1 genetic interval restored normal mobilization of cholesterol from the lysosomal compartment. Unexpectedly, the YAC-containing cell lines have overall cellular cholesterol concentrations that are comparable to wild-type levels, despite the assumed presence of the SCAP mutation. This phenotypic change results from a reduction in endogenous sterol synthesis, LDL receptor message, and HMG-CoA reductase message.

Genetic analysis of the SCAP gene revealed that the YAC-expressing CT60 cells have normal regulation of these sentinel cholesterogenic genes as a result of selective silencing of the mutant SCAP allele, which appears to be independent of functional NPC1 expression.

Abbreviations: ER, endoplasmic reticulum; NPC1, Niemann-Pick type C1; SCAP, sterol regulatory element binding protein cleavage-activating protein; SREBP, sterol regulatory element binding protein; SSD, sterol-sensing domain; YAC, yeast artificial chromosome

Supplementary key words cholesterol • sterol regulatory element binding protein • sterol regulatory element binding protein cleavage-activating protein • Chinese hamster ovary cells • Niemann-Pick type C1


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