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

• Main Authors: Jean Ann Maguire, Jerry W. Reagan, Jr.
• Format: Article
• Language: English
• Published: Elsevier 2005-09-01
• Series: Journal of Lipid Research
• Subjects: cholesterol; sterol regulatory element binding protein; sterol regulatory element binding protein cleavage-activating protein; Chinese hamster ovary cells; Niemann-Pick type C1
• Online Access: http://www.sciencedirect.com/science/article/pii/S0022227520329308

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.