Figueroa Bruno, Chen Sulin, Oyler George A, Hardwick J Marie, Betenbaugh Michael J
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Biotechnol Bioeng. 2004 Mar 20;85(6):589-600. doi: 10.1002/bit.10913.
A balance between proliferation and cell death is critical for achieving desirable high cell densities in mammalian cell culture. In this study, we evaluate a recently discovered anti-apoptotic gene, aven, and examine its effectiveness alone and in combination with a member of the Bcl-2 family, bcl-xL. The commercially popular cell line, Chinese hamster ovary (CHO), was genetically modified to constitutively express aven, bcl-xL, and the two genes in combination. Cells were exposed to several model insults that simulate severe bioreactor environments, including serum deprivation, spent medium, and Sindbis virus infection, as well as staurosporine, a known chemical inducer of apoptosis. CHO cells exhibited DNA fragmentation, a hallmark of apoptosis, after exposure to these model insults. After exposure to serum deprivation, 4- and 5-day spent medium, and staurosporine, cells expressing Aven provided limited protection against cell death when compared with the protection afforded by cells expressing Bcl-xL alone. However, the highest survival levels for all insults were achieved when Aven was expressed in combination with Bcl-xL. In fact, Aven appeared to act synergistically to enhance the protective function of Bcl-xL for several insults, because the protective function of the two genes expressed together in one cell line often exceeded the additive protective levels of each anti-apoptosis gene expressed alone. Surprisingly, Aven expression provided a mildly pro-apoptotic response in CHO isolates infected with Sindbis virus. However, CHO cells expressing both Bcl-xL and Aven showed protection against Sindbis virus infection due to the inhibitory properties of the bcl-xL anti-apoptosis gene. This study shows that combinatorial anti-apoptosis cell engineering strategies may be the most effective mechanisms for providing extended protection against cell death in mammalian cell culture.
在哺乳动物细胞培养中，增殖与细胞死亡之间的平衡对于实现理想的高细胞密度至关重要。在本研究中，我们评估了最近发现的抗凋亡基因aven，并单独以及与Bcl-2家族成员bcl-xL联合检测其有效性。对商业上常用的细胞系——中国仓鼠卵巢（CHO）细胞系进行基因改造，使其组成型表达aven、bcl-xL以及这两个基因的组合。将细胞暴露于几种模拟严重生物反应器环境的模型刺激因素下，包括血清剥夺、用过的培养基、辛德毕斯病毒感染，以及已知的凋亡化学诱导剂星形孢菌素。CHO细胞在暴露于这些模型刺激因素后出现了DNA片段化，这是凋亡的一个标志。在暴露于血清剥夺、4天和5天的用过的培养基以及星形孢菌素后，与单独表达Bcl-xL的细胞所提供的保护相比，表达Aven的细胞对细胞死亡的保护作用有限。然而，当Aven与Bcl-xL联合表达时，对所有刺激因素均实现了最高的存活水平。事实上，对于几种刺激因素，Aven似乎协同发挥作用以增强Bcl-xL的保护功能，因为在一个细胞系中共同表达的这两个基因的保护功能常常超过单独表达的每个抗凋亡基因的累加保护水平。令人惊讶的是，在感染辛德毕斯病毒的CHO分离株中，Aven的表达产生了轻度的促凋亡反应。然而，由于bcl-xL抗凋亡基因的抑制特性，同时表达Bcl-xL和Aven的CHO细胞对辛德毕斯病毒感染表现出保护作用。本研究表明，组合抗凋亡细胞工程策略可能是在哺乳动物细胞培养中提供延长的细胞死亡保护的最有效机制。
