Large-scale gene expression analysis of cholesterol dependence in NS0 cells.
NS0, a nonsecreting mouse myeloma cell, is a major host line used for recombinant antibody production. These cells have a cholesterol-dependent phenotype and rely on an exogenous supply of cholesterol for their survival and growth. To better understand the physiology underlying cholesterol dependence, we compared NS0 cells, cultivated under standard cholesterol-dependent growth conditions (NS0), to cells adapted to cholesterol-independent conditions (NS0 revertant, NS0_r). Large-scale transcriptional analyses were done using the Affymetrix GeneChip array, MG-U74Av2. The transcripts expressed differentially across the two cell lines were identified. Additionally, proteomic tools were employed to analyze cell lysates from these two cell lines. Cellular proteins from both NS0 and NS0_r were subjected to 2D gel electrophoresis. MALDI-TOF mass spectrometry was performed to determine the identity of the differentially expressed spots. We examined the expression level of mouse genes directly involved in cholesterol biosynthesis, lipid metabolism, and central energy metabolism. Most of these genes were downregulated in the revertant cell type, NS0_r, compared to NS0. Overall, a large number of genes are expressed differentially, indicating that the reversal of cholesterol dependency has a profound effect on cell physiology. It is probable that a single gene mutation, activation, or inactivation is responsible for cholesterol auxotrophy. However, the wide-ranging changes in gene expression point to the distinct possibility of a regulatory event affecting the reversibility of auxotrophy, either directly or indirectly.
Seth
Gargi
G
Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Ave. SE, Minneapolis, Minnesota 55455-0132, USA.
Philp
Robin J
RJ
Denoya
Claudio D
CD
McGrath
Katherine
K
Stutzman-Engwall
Kim J
KJ
Yap
Miranda
M
Hu
Wei-Shou
WS
D000818:Animals; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D051379:Mice; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism; D009101:Multiple Myeloma / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D020543:Proteome / Q000378:metabolism*; D015398:Signal Transduction
D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D020543:Proteome; D002784:Cholesterol