Reverting cholesterol auxotrophy of NS0 cells by altering epigenetic gene silencing

Abstract

NS0 is a cholesterol-requiring mouse myeloma cell line widely used in the production of recombinant antibodies. We have previously reported that the deficiency of 17beta-hydroxysteroid dehydrogenase type7 (Hsd17b7) is responsible for the cholesterol auxotrophy of NS0 cells. Here we demonstrate DNA methylation to be the mechanism underlying transcriptional suppression of Hsd17b7 in cholesterol dependent NS0 cells. Analysis of the DNA methylation pattern revealed methylation of the CpG-rich region upstream of the Hsd17b7 transcription start site in NS0 cells. This is in contrast to the unmethylated status of this sequence in a naturally isolated cholesterol independent revertant cell population (NS0_r). This transcriptional repression was relieved after treating cells with the demethylating drug, 5-azacytidine. Drug treatment also gave rise to high frequency cholesterol-independent variants. Characterization of revertants revealed substantially elevated transcript level of 17beta-hydroxysteroid dehydrogenase type7 (Hsd17b7) gene along with hypomethylation of the CpG-rich region. These results affirm that deficiency of Hsd17b7 causes cholesterol dependence of NS0 cells. Furthermore, induction of cholesterol independence by altering DNA methylation pattern alludes to the role of epigenetics in the metabolic adaptation of NS0 cells. With the widespread use of NS0 cells, this finding will have a significant impact on the optimization of recombinant antibody production processes.

Derivation and characterization of cholesterol‐independent non‐GS NS0 cell lines for production of recombinant antibodies

“…conditions isn't negligible, it is clearly achievable (Birch et al, 1994;Kawamoto et al, 1983;Keen, 1995;Seth et al, 2006b). Consistent with the findings of these other groups, our results indicate that only a small percentage of NS0 cells are capable of growing in protein-free, cholesterol-free medium, as evidenced by the sharp decrease in viability that was seen in medium containing only 0.3% FBS.…”

“…Recently, it has been reported that cholesterol auxotrophy in NS0 cells is due to reduced expression of the Hsd17b7 gene, encoding a 3-ketosteroid reductase (Seth et al, 2006a). Methylation upstream of the Hsd17b7 gene appears to be responsible for transcriptional suppression of this gene (Seth et al, 2006b). However, the underlying mechanism of cholesterol prototrophy in the NS0-PFCF cells described here has not been characterized.…”

Cell Culture Processes in Monoclonal Antibody Production

“…[10][11][12] NS0 cells lack endogenous glutamine synthetase (GS) enzyme activity making them suitable for use with GS as a selectable marker for recombinant antibody expression. 13 High antibody productivity has been reported from non-GS NS0 cell lines as well.…”

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

“…Two effects contribute: genomic variation between cell lines is observed [5], as well as what has been termed phenotypic drift: the slow but continuous changes and adaptations observed in cells maintained in culture for a prolonged period of time [15,16]. The latter can be caused by both genomic changes and epigenetic regulation [17][18][19]. This genomic and phenotypic heterogeneity has advantages and disadvantages: On the one hand it allows the effective selection of cells with different physiological and process relevant properties during screening [6,[20][21][22][23][24][25], on the other hand it limits the stability of these properties [21,26,27] and requires large numbers of subclones to be tested over prolonged periods of time to identify the few stable clones suited for production.…”