Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures

Yun, Chee Yong; Liu, Sen; Lim, Sing Fee; Wang, Tianhua; Chung, Beatrice Y.F.; Teo, Joong Jiat; Chuan, Kok Hwee; Soon, Allyson; Goh, Keng Siong; Song, Zhiwei

doi:10.1016/j.ymben.2007.06.001

Cited by 29 publications

(14 citation statements)

References 46 publications

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“…It is also necessary to identify the controlling mechanism of the heparin synthesis pathway, which is not fully understood. Moreover, while previous studies have explored CHO cell metabolism (Ahn and Antoniewicz, 2011; Nolan and Lee, 2011) and engineering CHO cells to enhance cell viability or improve recombinant protein glycosylation (Yun et al, 2007; Goh et al, 2010), there are no reports to date using metabolic engineering to produce non-protein products in CHO cells.…”

Section: Discussionmentioning

confidence: 99%

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

Baik

Gasimli

Yang

et al. 2012

Metabolic Engineering

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Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS / heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS / heparin biosynthesis might be necessary.

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Section: Discussionmentioning

confidence: 99%

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

Baik

Gasimli

Yang

et al. 2012

Metabolic Engineering

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“…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.…”

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confidence: 99%

“…14,15 Also, reports on downregulation of caspases have shown to inhibit the execution of apoptosis. 16,17 In addition, Lim et al 18 show that pro-apoptotic Bcl-2 family proteins such as Bax and BAK when down-regulated by siRNA result in the increase of viability during fed-batch culture.…”

mentioning

confidence: 99%

Autophagy and apoptosis in Chinese hamster ovary cell culture

Hwang

Lee²

2008

Autophagy

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“…Until now, apoptosis in rCHO cell culture has been recognized as an important task to be solved, and therefore, there have been numerous studies focusing on the onset of apoptosis and anti-apoptotic gene engineering in rCHO cells (Arden and Betenbaugh, 2004). As a result, it was found that the onset of apoptosis in rCHO cells can be delayed by promoting anti-apoptotic factors as well as by inhibiting the expression of pro-apoptotic factors (Chiang and Sisk, 2005;Figueroa et al, 2007;Kim and Lee, 2000;Lim et al, 2006;Sauerwald et al, 2003;Sung et al, 2007;Tey et al, 2000;Wong et al, 2006;Yun et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Effect of Bcl‐x_L overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition

Kim

Mohan

et al. 2009

Biotech & Bioengineering

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Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl-x(L) overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)-producing rCHO cell line with regulated Bcl-x(L) overexpression (EPO-off-Bcl-x(L)) was established using the Tet-off system. The expression level of Bcl-x(L) in EPO-off-Bcl-x(L) cells was tightly regulated by doxycycline in a dose-dependent manner. Bcl-x(L) overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl-x(L) overexpression suppressed apoptosis by inhibiting the activation of caspase-3 and -7. Simultaneously, Bcl-x(L) overexpression also delayed autophagy, characterized by LC3-II accumulation. Immunoprecipitation analysis with a Flag-tagged Bcl-x(L) revealed that Bcl-x(L) interacts with Bax and Bak, essential mediators of caspase-dependent apoptosis, as well as with Beclin-1, an essential mediator of autophagy, and may inhibit their pro-cell death function. Taken together, it was found that Bcl-x(L) overexpression inhibits both apoptosis and autophagy in rCHO cell culture.

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Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures

Cited by 29 publications

References 46 publications

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

Autophagy and apoptosis in Chinese hamster ovary cell culture

Effect of Bcl‐x_L overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition

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Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures

Cited by 29 publications

References 46 publications

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

Autophagy and apoptosis in Chinese hamster ovary cell culture

Effect of Bcl‐xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition

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Product

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Effect of Bcl‐x_L overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition