Overexpression ofbcl-2 inhibits sodium butyrate-induced apoptosis in Chinese hamster ovary cells resulting in enhanced humanized antibody production

Abstract

Sodium butyrate (NaBu) can enhance the expression of genes from some of the mammalian promoters including cytomegalovirus (CMV) and simian virus 40 (SV40), but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, a survival protein, human Bcl-2, was overexpressed in recombinant Chinese hamster ovary (CHO) cells (SH2-0.32), producing a humanized antibody directed against the S surface antigen of hepatitis B virus. When batch cultures of both control cells transfected with bcl-2-deficient plasmid (SH2-0.32-⌬bcl-2) and cells transfected with bcl-2 expression plasmid (14C6-bcl-2) were performed in the absence of NaBu, both cells showed similar profiles of cell viability and antibody production. Compared with the SH2-0.32-⌬bcl-2 culture, under the condition of NaBu addition at the exponential growth phase, overexpression of the bcl-2 gene considerably suppressed the NaBuinduced apoptosis of 14C6-bcl-2 by inhibiting caspase 3 activity and extending culture longevity by >2 days. As a result, the final antibody concentration of 14C6-bcl-2 culture was twofold higher than that of SH2-0.32-⌬bcl-2 culture in the presence of NaBu and threefold higher than that of SH2-0.32-⌬bcl-2 and 14C6-bcl-2 cultures in the absence of NaBu.

Autophagy and apoptosis in Chinese hamster ovary cell culture

“…Numerous studies have shown that hyperosmolality or addition of sodium butyrate (NaBu) often causes stressful conditions, while enhancing the overall productivity of protein expression. 6,7 It was reported that NaBu induces apoptotic cell death 8 and arrests cells at the G 1 phase of the cell cycle. 9 Biopharmaceutical companies have become a multi billion-dollar industry, generating various recombinant proteins.…”

“…Up to now, there have been many approaches to overcome apoptotic cell death during batch cultures by (1) genetically modifying important genes in cell growth, 8,[10][11][12][13][14][15][16][17][18] (2) supplementing essential nutrients, [19][20][21] or (3) adding chemical inhibitors. 22,23 We address each of these approaches below.…”

“…A number of studies show that upregulating pro-survival Bcl-2 family proteins protects cells from apoptotic death caused by diverse culture stresses, by regulating the mitochondrial membrane potential. 8,[10][11][12] Moreover, manipulation of genes that are important for metabolism such as the fructose transporter also increase cell survival. 13 In addition, an increasing number of reports support the view that engineering pro-survival genes such as E1B-19K, Aven, Fadd, and Faim, is a potential method to achieve higher cell viability.…”

Modulation of heparan sulfate biosynthesis by sodium butyrate in recombinant CHO cells

“…Butyrate treatment in CHO cells affects cellular processes such as cytoskeleton reorganization, protein transport, nucleosome assembly, nucleotide metabolism, glycosylation, protein folding, oxidative stress responses, lipid metabolism, cholesterol biosynthesis, glycolysis, cell cycle progression, and apoptosis (Yee et al 2008;De Leon et al 2007). Butyrate has been used in cell engineering strategies, and butyrate treatment in CHO cells often increases recombinant protein expression (Chun et al 2003;De Leon et al 2007;Hendrick et al 2001;Kim and Lee 2000;Chotigeat et al 1994;Mimura et al 2001;Wang et al 2002;Jiang and Sharfstein 2008). The current work explores the effect of butyrate on glycosaminoglycan (GAG) profiles in recombinant CHO cells in our efforts to produce a bioengineered heparin (HP).…”

“…Sodium butyrate (CH 3 CH 2 CH 2 CO 2 -) is a histone deacetylase inhibitor that modulates gene expression of cellular processes, particularly transgene expression in Chinese hamster ovary (CHO) cells (Chotigeat et al 1994;Chun et al 2003;De Leon et al 2007;Hendrick et al 2001;Jiang and Sharfstein 2008;Kim and Lee 2000;Mimura et al 2001;Wang et al 2002).…”