Apoptosis in batch cultures of Chinese Hamster Ovary cells

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cell s continues to be the inability to maintain the viability of the cultures o ver an extended period of time. The rapid decline in viability at the end o f the culture is exacerbated by the absence of serum. In trying to reduce t he extent of death in these cultures, we first tried to determine the mode of death. We found that more than 80% of the cells in a standard serum-free batch culture of CHO cells in suspension died via apoptosis-as evidenced b y condensed chromatin and the appearance of a characteristic DNA ladder. Fu rthermore, when protein synthesis was inhibited using cycloheximide, the ce lls underwent rapid apoptosis indicating that death proteins were present i n greater abundance than survival proteins in our CHO cells. Cell lysate fr om CHO cells showed evidence of cysteine protease (caspase) activity. Caspa ses of the Interleukin-1-beta-Converting Enzyme (ICE) family, e.g., CPP32, Mch-1, etc., have been implicated in the apoptotic process. Surprisingly, a caspase peptide inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoro-methyl-k etone (z-VAD.fmk), was unable to substantially extend the life of a serum-f ree batch culture of CHO cells. In addition, z-VAD.fmk was only marginally able to extend viability in response to withdrawal of growth and survival f actors, insulin and transferrin. In both these instances, z-VAD.fmk was abl e to prevent cleavage of caspase substrates, but not protect cells from dea th. However, we found that bcl-2 expression was able to significantly exten d viabilities in CHO batch culture. Bcl-2 expression also substantially ext ended the viability of cultures in response to insulin and transferrin with drawal. These results provide interesting insights into the pathways of dea th in a CHO cell. (C) 1999 John Wiley & Sons, Inc.