title: Anti-cell death engineering of CHO cells: co-overexpression of Bcl-2 for apoptosis inhibition, Beclin-1 for autophagy induction.
abstract: 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.
authors: Department of Biological Sciences and Graduate School of Nanoscience and Technology (WCU), KAIST, 335 Gwahak-Ro, Yuseong-Gu, Daejeon 305-701, Republic of Korea.
authors: Jae Seong
authors: JS
authors: Lee
authors: Tae Kwang
authors: TK
authors: Ha
authors: Jin Hyoung
authors: JH
authors: Park
authors: Gyun Min
authors: GM
authors: Lee
mesh_terms: D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis* / Q000235:genetics; D001343:Autophagy; D001709:Biotechnology / Q000379:methods*; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival; D003412:Cricetulus; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D051017:Apoptosis Regulatory Proteins; D019253:Proto-Oncogene Proteins c-bcl-2