Mastrangelo A J, Zou S, Hardwick J M, Betenbaugh M J
Department of Chemical Engineering, The Johns Hopkins University, 028 New Engineering Building, 3400 North Charles Street, Baltimore, Maryland 21218-2694, USA.
Biotechnol Bioeng. 1999 Nov 5;65(3):298-305.
Viral expression systems allow for the rapid production of large amounts of recombinant protein in cell culture. In particular, Sindbis virus vectors now exist that make possible the expression of a variety of heterologous proteins in mammalian culture systems. Unfortunately, infection of cultured cells with Sindbis virus vectors typically results in apoptotic cell death, as demonstrated in the current study by DNA laddering and fluorescence microscopy. Fortunately, it has recently been demonstrated that apoptosis can be inhibited in vitro by certain chemical reagents that are capable of blocking specific steps during the cell death cascade. In this study, a rat prostate carcinomal cell line, AT3-neo, was infected with a Sindbis virus vector containing the gene for chloramphenicol acetyltransferase (dsSV-CAT) in the presence of several representative antiapoptotic chemicals and analyzed for cell viability as well as recombinant protein production. N-acetylcysteine (NAC), pyrrolidine dithiocarbamate (PDTC), bongkrekic acid (BA), and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) all exhibited the capacity to limit apoptosis in the infected cells. In fact, after just 1 day, percentage viabilities of the cells exposed to chemical reagents were between 72% and 91%, compared with 44% for the untreated controls. Furthermore, cells maintained on these agents were able to survive the infection from 1 to 3 days longer than the control samples. In addition to providing gains in cell viability, chemical treatment allowed for higher levels of recombinant protein production in most cases. Maximum chloramphenicol acetyltransferase (CAT) productivities in cells maintained on BA, NAC, and Z-VAD.fmk were 1.7-, 2.2-, and 3.9-fold higher than those obtained from the untreated cultures. Consequently, the addition of chemical reagents to culture media as a means of inhibiting apoptosis may be a valuable tool in the cell culture industry, where cell death severely limits productivity levels and adds significantly to production costs.
病毒表达系统能够在细胞培养中快速大量生产重组蛋白。特别是，目前存在辛德毕斯病毒载体，使得在哺乳动物培养系统中表达多种异源蛋白成为可能。不幸的是，正如本研究通过DNA梯状条带分析和荧光显微镜所证明的那样，用辛德毕斯病毒载体感染培养细胞通常会导致凋亡性细胞死亡。幸运的是，最近已证明，某些能够阻断细胞死亡级联反应中特定步骤的化学试剂可在体外抑制凋亡。在本研究中，在几种代表性的抗凋亡化学试剂存在的情况下，用含有氯霉素乙酰转移酶基因（dsSV-CAT）的辛德毕斯病毒载体感染大鼠前列腺癌细胞系AT3-neo，并分析细胞活力以及重组蛋白的产生。N-乙酰半胱氨酸（NAC）、吡咯烷二硫代氨基甲酸盐（PDTC）、硼酸（BA）和N-苄氧羰基-Val-Ala-Asp-氟甲基酮（Z-VAD.fmk）均表现出限制感染细胞凋亡的能力。事实上，仅1天后，暴露于化学试剂的细胞的活力百分比在72%至91%之间，而未处理对照的细胞活力百分比为44%。此外，维持在这些试剂上的细胞能够比对照样品多存活1至3天。除了提高细胞活力外，化学处理在大多数情况下还能实现更高水平的重组蛋白产生。维持在BA、NAC和Z-VAD.fmk上的细胞中氯霉素乙酰转移酶（CAT）的最大生产力分别比未处理培养物中获得的生产力高1.7倍、2.2倍和3.9倍。因此，向培养基中添加化学试剂作为抑制凋亡的手段可能是细胞培养行业中的一种有价值的工具，在该行业中，细胞死亡严重限制了生产力水平并显著增加了生产成本。
