Submitted on May 17, 2005
Revised on June 15, 2005
Accepted on June 27, 2005
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
Pathology Dept., Wake Forest University School of Medicine, Winston-Salem, NC 27157
Corresponding Author: jreagan{at}wfubmc.edu
The Sterol Regulatory Element Binding Protein/SREBP Cleavage Activating Protein (SREBP/SCAP) complex regulates 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, the 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 (Gu, J.Z. et al. 1997 PNAS). 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 due to selective silencing of the mutant SCAP allele, which appears to be independent of functional NPC1 expression.
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