Inhibiting the apoptosis pathway using MDM2 in mammalian cell cultures.
The ability to regulate apoptosis in mammalian cell cultures represents one approach to developing more economical and efficient processes. Genetic modification of cells using anti-apoptotic genes is one method that may be used to improve cellular performance. This study investigates a method to inhibit upstream apoptosis pathways through the overexpression of MDM2, an E3 ubiquitin ligase for p53. Both 293 and CHO cells expressing MDM2 were examined under both batch and spent media conditions. For batch cultures, MDM2 overexpression increased viable cell densities and viabilities over control cells with the largest enhancements observed in CHO cells. When CHO cells were passaged without medium exchange, cells expressing MDM2 reached a viable cell density that was nearly double the control and survived for an extra day in culture. When exposed to spent media initially, both 293-MDM2 and CHO-MDM2 cells continued to grow for 2 days while the control cells stopped growing after the first day. DNA analysis using flow cytometry confirmed that while CHO controls were found to be undergoing DNA fragmentation, CHO-MDM2 cells exhibit DNA degradation at a much slower rate. When compared to Bcl-2-expressing cells, MDM2 expression showed greater protection against apoptosis in passaged culture, spent medium, and following transient p53 overexpression. However, expression of the RING sequence of MDM2 responsible for E3 ligase activity without the other components of the protein was found to be toxic to 293 cells in culture. These results suggest that the overexpression of heterologous MDM2 represents a promising method to delay apoptosis in mammalian cell cultures.
Arden
Nilou
N
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Majors
Brian S
BS
Ahn
Shin-Hyung
SH
Oyler
George
G
Betenbaugh
Michael J
MJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D008322:Mammals; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry / Q000494:pharmacology*
C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2