Sauerwald Tina M, Oyler George A, Betenbaugh Michael J
Department of Chemical Engineering, The Johns Hopkins University, 3400 North Charles St., Baltimore, Maryland 21218, USA.
Biotechnol Bioeng. 2003 Feb 5;81(3):329-40. doi: 10.1002/bit.10473.
Apoptosis in mammalian cell culture is associated with decreased bioproduct yields and can be inhibited through altering the intracellular signaling pathways mediating programmed cell death. In this study, we evaluated the capacity to inhibit caspases to maintain high viable cell numbers in CHO and 293 cultures. Two genetic caspase inhibitors, XIAP and CrmA, were examined along with a mutant of each, XIAP-BIR123NC, which contains three BIR domains but lacks the RING finger, and CrmA-DQMD, which has CrmA's pseudosubstrate site replaced with that of another caspase inhibitor, p35. Stable CHO pooled and 293 clonal cell lines expressing each protein were exposed to apoptotic insults, including spent medium, Sindbis virus, and etoposide. For each insult the mutated protein resulted in higher viabilities than its wild-type counterpart. However, the mutants provided different levels of protection, depending on the insult considered. CrmA-DQMD was the preferred inhibitor for spent medium-induced apoptosis, whereas XIAP-BIR123NC conferred better protection for etoposide-induced death. Addition of Z-VAD.fmk to the genetically engineered cells enhanced viabilities in the presence of spent medium or etoposide; however, the largest increases in viability were experienced by the control cells, indicating an overlap in caspase inhibition between the genetic and chemical inhibitors. Finally, parental 293 cells were treated with caspase-8 and -9 inhibitors, Z-IETD.fmk and Z-LEHD.fmk, in concert with spent medium or etoposide exposure. Spent medium-induced death was delayed more readily with the caspase-8 inhibitors, CrmA-DQMD and Z-IETD.fmk, and etoposide-induced death was stalled more so with XIAP-BIR123NC and Z-LEHD.fmk. These results suggest that the apoptosis pathways induced and the level of protection afforded by a particular caspase inhibitor may vary with the insult considered.
哺乳动物细胞培养中的细胞凋亡与生物制品产量降低相关，并且可以通过改变介导程序性细胞死亡的细胞内信号通路来抑制。在本研究中，我们评估了抑制半胱天冬酶以在CHO和293细胞培养物中维持高活细胞数的能力。研究了两种遗传半胱天冬酶抑制剂XIAP和CrmA，以及它们各自的一个突变体，XIAP-BIR123NC（其包含三个BIR结构域但缺乏指环结构）和CrmA-DQMD（其将CrmA的假底物位点替换为另一种半胱天冬酶抑制剂p35的假底物位点）。将表达每种蛋白质的稳定CHO混合细胞系和293克隆细胞系暴露于凋亡刺激，包括用过的培养基、辛德毕斯病毒和依托泊苷。对于每种刺激，突变蛋白比其野生型对应物具有更高的活力。然而，根据所考虑的刺激，突变体提供了不同水平的保护。CrmA-DQMD是用过的培养基诱导的细胞凋亡的首选抑制剂，而XIAP-BIR123NC对依托泊苷诱导的细胞死亡提供了更好的保护。在存在用过的培养基或依托泊苷的情况下，向基因工程细胞中添加Z-VAD.fmk可提高活力；然而，对照细胞的活力增加最大，这表明遗传抑制剂和化学抑制剂在半胱天冬酶抑制方面存在重叠。最后，将亲本293细胞与用过的培养基或依托泊苷暴露同时用半胱天冬酶-8和-9抑制剂Z-IETD.fmk和Z-LEHD.fmk处理。用过的培养基诱导的细胞死亡更容易被半胱天冬酶-8抑制剂CrmA-DQMD和Z-IETD.fmk延迟，而依托泊苷诱导的细胞死亡更多地被XIAP-BIR123NC和Z-LEHD.fmk阻止。这些结果表明，所诱导的凋亡途径以及特定半胱天冬酶抑制剂提供的保护水平可能因所考虑的刺激而异。
