Apoptosis in batch cultures of Chinese hamster ovary cells

Biotechnol Bioeng. 1999 Mar 20;62(6):632-40. doi: 10.1002/(sici)1097-0290(19990320)62:6<632::aid-bit2>3.0.co;2-i.

Abstract

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the 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 by condensed chromatin and the appearance of a characteristic DNA ladder. Furthermore, when protein synthesis was inhibited using cycloheximide, the cells underwent rapid apoptosis indicating that death proteins were present in greater abundance than survival proteins in our CHO cells. Cell lysate from CHO cells showed evidence of cysteine protease (caspase) activity. Caspases 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-ketone (z-VAD.fmk), was unable to substantially extend the life of a serum-free 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 factors, insulin and transferrin. In both these instances, z-VAD.fmk was able to prevent cleavage of caspase substrates, but not protect cells from death. However, we found that bcl-2 expression was able to significantly extend viabilities in CHO batch culture. Bcl-2 expression also substantially extended the viability of cultures in response to insulin and transferrin withdrawal. These results provide interesting insights into the pathways of death in a CHO cell.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Chloromethyl Ketones / pharmacology
  • Animals
  • Apoptosis / drug effects*
  • Apoptosis / genetics*
  • CHO Cells / cytology
  • CHO Cells / drug effects*
  • CHO Cells / pathology
  • Caspase 3
  • Caspases / analysis
  • Caspases / drug effects
  • Caspases / metabolism
  • Cell Division / drug effects
  • Cell Division / genetics
  • Cell Survival / drug effects
  • Cell Survival / genetics
  • Cells, Cultured
  • Cricetinae
  • Culture Media, Serum-Free
  • Cycloheximide / pharmacology
  • Cysteine Proteinase Inhibitors / pharmacology
  • Dose-Response Relationship, Drug
  • Humans
  • Protein Synthesis Inhibitors / pharmacology
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Transfection

Substances

  • Amino Acid Chloromethyl Ketones
  • Culture Media, Serum-Free
  • Cysteine Proteinase Inhibitors
  • Protein Synthesis Inhibitors
  • Proto-Oncogene Proteins c-bcl-2
  • benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
  • Cycloheximide
  • CASP3 protein, human
  • Caspase 3
  • Caspases