Kim Yeon-Gu, Kim Jee Yon, Mohan Chaya, Lee Gyun Min
Department of Biological Sciences, KAIST, 373-1 Kusong-Dong, Yusong-Gu, Daejon, Korea.
Biotechnol Bioeng. 2009 Jul 1;103(4):757-66. doi: 10.1002/bit.22298.
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.
在培养过程中营养物质缺乏时，重组中国仓鼠卵巢（rCHO）细胞会经历两种程序性细胞死亡（PCD），即凋亡和自噬。为了研究Bcl-x(L)过表达对rCHO细胞凋亡和自噬的影响，利用Tet-off系统建立了一种产生促红细胞生成素（EPO）且Bcl-x(L)过表达受调控的rCHO细胞系（EPO-off-Bcl-x(L)）。EPO-off-Bcl-x(L)细胞中Bcl-x(L)的表达水平受强力霉素严格调控，呈剂量依赖性。在分批培养中，Bcl-x(L)过表达增强了细胞活力并延长了培养寿命。在分批培养后期营养物质耗尽时，Bcl-x(L)过表达通过抑制caspase-3和-7的激活来抑制凋亡。同时，Bcl-x(L)过表达也延迟了自噬，其特征是LC3-II积累。用Flag标记的Bcl-x(L)进行免疫沉淀分析表明，Bcl-x(L)与caspase依赖性凋亡的关键介质Bax和Bak相互作用，也与自噬的关键介质Beclin-1相互作用，并可能抑制它们的促细胞死亡功能。综上所述，发现Bcl-x(L)过表达在rCHO细胞培养中同时抑制凋亡和自噬。
