Large‐scale gene expression analysis of cholesterol dependence in NS0 cells

Abstract

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.

Production of Retroviral and Lentiviral Gene Therapy Vectors: Challenges in the Manufacturing of Lipid Enveloped Virus

“…Unless other supplements are added, serum is the only lipid source of the culture medium and, although cells should be able to sense lipid absence in the culture medium and activate biosynthetic pathways to stand up to lipid deprivation, the activation of lipid de novo synthesis may take hours or days, depending on the cell type (Alberts et al 1974;Spector et al 1980). In some cases, cells can no longer synthesize certain lipids (Seth et al 2005). Membrane lipids are active players in the complex process of retroviral assembling, and pseudotyping that takes place at the host cell membrane, in which interactions of membrane lipid rafts select both envelope and core proteins, recruiting later the other viral components by cooperative interaction.…”

Using cell engineering and omic tools for the improvement of cell culture processes

“…The first combined transcriptomic and proteomic analysis was accomplished on a NS0 cell line by Seth et al (2005). In this study, the gene and protein expression profile of NS0 cholesteroldependent phenotype, cultivated under standard cholesterol-dependent growth conditions (NS0) was compared to cells adapted to cholesterolindependent conditions (NS0 revertant) (Seth et al 2005).…”

“…In this study, the gene and protein expression profile of NS0 cholesteroldependent phenotype, cultivated under standard cholesterol-dependent growth conditions (NS0) was compared to cells adapted to cholesterolindependent conditions (NS0 revertant) (Seth et al 2005). Overall, a large number of genes were expressed differentially and mostly downregulated in the cholesterol biosynthesis, lipid metabolism and central energy metabolism.…”

Proteomic profiling of recombinant cells from large-scale mammalian cell culture processes

“…Proteomic studies have also been performed to gain a better understanding of the molecular mechanisms that regulate cholesterol-dependent growth in NS0 cells (Seth et al 2005). NS0 cells by their nature are cholesterol-dependent, thus their growth is dependent on the provision of cholesterol to the medium; however this presents difficulties because of its poor solubility.…”

“…Adaptation generally involves passaging cells over time during which the cholesterol concentration is gradually reduced (Sinacore et al 1996). In this study by Seth et al (2005), proteomic tools were used to compare the protein profiles of regular 'wild-type' NS0 cells and a 'revertant' cell line that can grow without cholesterol. 2D-PAGE and mass spectrometry identified 23 proteins that were differentially regulated in the revertant cells.…”