Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-x(L) on productivity, cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media.
We have engineered dihydrofolate reductase-negative (dhfr-/-) Chinese hamster ovary (CHO) DG44 cells adapted for growth in serum-free suspension cultures for simultaneous expression of the common cold therapeutic, the soluble intercellular adhesion molecule 1 (sICAM), and the antiapoptosis determinants bcl-2 or bcl-x(L). Detailed analyses of titer and antiapoptosis characteristics of these production cell lines included an independent (sICAM; bcl-2/bcl-x(L)) as well as a cocistronic (sICAM-(bcl-2/bcl-x(L))) expression set-up in which translation-initiation of the survival cistron is driven by an internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). In transient transfections or stable mixed populations and in comparison to isogenic sICAM-only control vectors, both bcl-x(L)-encoding configurations achieved higher sICAM yields while bcl-2 over-expression resulted in decreased product levels. Overall, the death-protective impact of bcl-2 and bcl-x(L) in engineered CHO-DG44 was not significant under typical batch-mode operation, an observation that was confirmed by clonal analysis. bcl-2 and bcl-x(L) displayed their antiapoptosis potential only following dhfr-based amplification in sICAM-producing CHO-DG44 cell lines. In all cases, bcl-x(L) outperformed bcl-2 in its cell death-protective capacity. Amplification-dependent high-level expression of mitochondria-localized bcl-2 family members required for successful antiapoptosis engineering may be essential to compensate for increased mitochondria numbers found to be associated with production cell lines grown in serum-free medium.
Meents
Heiko
H
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Zurich, CH-8093 Zurich, Switzerland.
Enenkel
Barbara
B
Eppenberger
Hans M
HM
Werner
Rolf G
RG
Fussenegger
Martin
M
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D016466:CHO Cells / Q000166:cytology / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D003001:Cloning, Molecular; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005784:Gene Amplification; D005786:Gene Expression Regulation; D024861:Genetic Enhancement / Q000379:methods*; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013535:Suspensions / Q000494:pharmacology; D014162:Transfection / Q000379:methods
D016895:Culture Media, Serum-Free; D019253:Proto-Oncogene Proteins c-bcl-2; D013535:Suspensions; D018799:Intercellular Adhesion Molecule-1