Reverting cholesterol auxotrophy of NS0 cells by altering epigenetic gene silencing.
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.
Seth
Gargi
G
Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Avenue SE, Minneapolis, Minnesota, USA.
Ozturk
Mustafa
M
Hu
Wei-Shou
WS
D015067:17-Hydroxysteroid Dehydrogenases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D051379:Mice; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000737:chemistry
D000964:Antimetabolites, Antineoplastic; D013447:Sulfites; D004247:DNA; D002784:Cholesterol; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase; D001374:Azacitidine; C042345:hydrogen sulfite