Large-Scale Gene Expression Analysis of Cholesterol Dependence in NS0 Cells Gargi Seth, 1 Robin J. Philp, 2 Claudio D. Denoya, 3 Katherine McGrath, 3 Kim J. Stutzman-Engwall, 3 Miranda Yap, 2 Wei-Shou Hu 1 1 Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Ave. SE, Minneapolis, Minnesota 55455-0132; e-mail: acre @cems.umn.edu 2 Bioprocessing Technology Institute, Singapore 3 Pfizer Global R&D, Pfizer Inc., Groton, Connecticut Received 5 August 2004; accepted 3 December 2004 Published online 13 April 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20429 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 Affy- metrix 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. B 2005 Wiley Periodicals, Inc. Keywords: NS0; mouse myeloma; cholesterol auxotroph; DNA microarray; gene expression; proteomics INTRODUCTION In the past decade, recombinant antibodies have become widely used as therapeutics for a number of major diseases (Chu and Robinson, 2001; Geisse et al., 1996; Verma et al., 1998). The primary host mammalian cells for antibody production are Chinese hamster ovary cells and mouse myeloma cells, including NS0 and Sp2/0 cells. NS0 are nonimmunoglobulin-secreting mouse myeloma cells. They are frequently used as efficient fusion partners or as host cells for transfecting heterologous immunoglobulin genes (Barnes et al., 2000; Bebbington et al., 1992). Being natural secretors of antibody molecules, a high titer antibody production has been reported from these cells (Dempsey et al., 2003; Sauer et al., 2000; Zhou et al., 1996, 1997). Since NS0 cells are widely used for the production of therapeutic and diagnostic proteins, significant prog- ress has been made to enhance their robustness and pro- ductivity via bioprocess optimization (Baker et al., 2001; deZengotita et al., 2002; Osman et al., 2002) and through cell engineering (Ibarra et al., 2003; Lasunskaia et al., 2003; Watanabe et al., 2002). In recent years the production of therapeutic biologics has seen increasing efforts to cultivate mammalian cells in chemically defined media and in the absence of animal- derived components (Battista et al., 1994; Gorfien et al., 1999, 2000; Hesse et al., 2003; Keen, 1995; Keen and Hale, 1996; Keen and Rapson, 1995; Keen and Steward, 1995). For serum free cultivation, the cell culture medium is of- ten supplemented with lipids (derived from plant or syn- thetic sources) in addition to other protein supplements. The effect of lipid supplementation on the physiology of hybridomas has been reported in the literature (Butler and Huzel, 1995; Savonniere et al., 1996). Lipids are important cellular constituents and are an important component in cell culture medium. Lipids constitute 2–10 mg/ml of serum, comprising of fatty acids (0.1–1.0 AM), phospholipids (0.7– 3.0 mg/ml) and cholesterol (10 AM) (Freshney, 2000). It is possible to cultivate some cells in a defined medium without supplementation of any lipids (Europa et al., 2000; Gambhir et al., 1999). However, NS0 cells are naturally cholesterol-dependent; not only is their growth greatly facilitated by lipid supplementation, but is also dependent on provision of cholesterol. The low solubility of B 2005 Wiley Periodicals, Inc. Correspondence to: Wei-Shou Hu Contract grant sponsor: Pfizer (to W.S.H.)