Proteomics analysis of chinese hamster ovary cells undergoing apoptosis during prolonged cultivation

Abstract

The degradation of environmental conditions, such as nutrient depletion and accumulation of toxic waste products over time, often lead to premature apoptotic cell death in mammalian cell cultures and suboptimal protein yield. Although apoptosis has been extensively researched, the changes in the whole cell proteome during prolonged cultivation, where apoptosis is a major mode of cell death, have not been examined. To our knowledge, the work presented here is the first whole cell proteome analysis of non-induced apoptosis in mammalian cells. Flow cytometry analyses of various activated caspases demonstrated the onset of apoptosis in Chinese hamster ovary cells during prolonged cultivation was primarily through the intrinsic pathway. Differential in gel electrophoresis proteomic study comparing protein samples collected during cultivation resulted in the identification of 40 differentially expressed proteins, including four cytoskeletal proteins, ten chaperone and folding proteins, seven metabolic enzymes and seven other proteins of varied functions. The induction of seven ER chaperones and foldases is a solid indication of the onset of the unfolded protein response, which is triggered by cellular and ER stresses, many of which occur during prolonged batch cultures. In addition, the upregulation of six glycolytic enzymes and another metabolic protein emphasizes that a change in the energy metabolism likely occurred as culture conditions degraded and apoptosis advanced. By identifying the intracellular changes during cultivation, this study provides a foundation for optimizing cell line-specific cultivation processes, prolonging longevity and maximizing protein production.

Fig. 1

Average cell growth and viability trend in run A and run B cultures

Fig. 2

Apoptosis progression in run A and run B based on FCM caspase 3/7 assay. FCM assays were performed for two additional time points in run B

Fig. 3

Comparison between cell subpopulations based on three FCM caspases assays of run B cultures. a C−PI−, b C+PI−, c C+PI+, d C−PI+ subpopulations. Caspase 8 and 9 are pathway-specific initiator caspases while caspase 3 is the common executioner caspases in different apoptotic pathways. Abbreviations: C caspase, PI propidium iodide

Fig. 4

MS identified differentially expressed protein spots from the comparison of healthy (day 2.5) to aging/apoptotic (day 8.5) CHO protein samples. Dotted line indicates proteins that upregulated over time while solid line indicates a reduction in expression. Estimates of molecular weight and PI were obtained from the protein identifications. Abbreviations: ATPB ATP synthase beta subunit, BiP binding immunoglobulin protein, ERp60 endoplasmic reticulum protein 60 kDa, FKBP52 FK506-binding protein 52, GAPDH glyceraldehyde-3-phosphate dehydrogenase, GRP gluose-regulated protein, HSC70 heat shock cognate protein 70 kDa, HSP heat shock protein, LAMR1 laminin receptor 1, LDH L-lactate dehydrogenase chain, PDI protein disulfide isomerase, PGM phosphoglycerate mutase, PGK phosphoglycerate kinase 1, PK pyruvate kinase isozyme M1/M2, Prx6 peroxiredoxin-6, TPI triosephosphate isomerase