Anti-cell death engineering of CHO cells: Co-overexpression of Bcl-2 for apoptosis inhibition, Beclin-1 for autophagy induction

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Genetic engineering approaches to inhibit cell death in Chinese hamster ovary (CHO) cell cultures have been limited primarily to anti-apoptosis engineering. Recently, autophagy has received attention as a new anti-cell death engineering target in addition to apoptosis. In order to achieve a more efficient protection of cells from the stressful culture conditions, the simultaneous targeting of anti-apoptosis and pro-autophagy in CHO cells (DG44) was attempted by co-overexpressing an anti-apoptotic protein, Bcl-2, and a key regulator of autophagy pathway, Beclin-1, respectively. Co-overexpression of Bcl-2 and Beclin-1 exhibited a longer culture period as well as higher viability during serum-free suspension culture, compared with the control (without co-overexpression of Bcl-2 and Beclin-1) and Bcl-2 overexpression only. In addition to the efficient inhibition of apoptosis by Bcl-2 overexpression, Beclin-1 overexpression successfully induced the increase in the autophagic marker protein, LC3-II, and autophagosome formation with the decrease in mTOR activity. Co-immunoprecipitation and qRT-PCR experiments revealed that the enforced expression of Beclin-1 increased Ulk1 expression and level of free-Beclin-1 that did not bind to the Bcl-2 despite the Bcl-2 overexpression. Under other stressful culture conditions such as treatment with sodium butyrate and hyperosmolality, co-overexpression of Bcl-2 and Beclin-1 also protected the cells from cell death more efficiently than Bcl-2 overexpression only, implying the potential of autophagy induction. Taken together, the data obtained here provide the evidence that pro-autophagy engineering together with anti-apoptosis engineering yields a synergistic effect and successfully enhances the anti-cell death engineering of CHO cells. Biotechnol. Bioeng. 2013; 110: 2195-2207. (c) 2013 Wiley Periodicals, Inc.
Publisher
WILEY-BLACKWELL
Issue Date
2013-08
Language
English
Article Type
Article
Keywords

HAMSTER OVARY CELLS; REGULATES AUTOPHAGY; SODIUM-BUTYRATE; CULTURE; PATHWAY; GENES; BATCH; MACROAUTOPHAGY; METABOLISM; EXPRESSION

Citation

BIOTECHNOLOGY AND BIOENGINEERING, v.110, no.8, pp.2195 - 2207

ISSN
0006-3592
DOI
10.1002/bit.24879
URI
http://hdl.handle.net/10203/175061
Appears in Collection
BS-Journal Papers(저널논문)
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