A comparison of the properties of a Bcl-xL variant to the wild-type anti-apoptosis inhibitor in mammalian cell cultures.
The overexpression of bcl-2 and its homologues is a widely used strategy to inhibit apoptosis in mammalian cell culture systems. In this study, we have evaluated the Bcl-2 homologue, Bcl-x(L) and compared its effectiveness to a Bcl-x(L) mutant lacking most of the non-conserved unstructured loop domain, Bcl-x(L)Delta (deletion of amino acids 26 through 83). The cell line, Chinese hamster ovary (CHO), was genetically modified to express constitutively Bcl-x(L) or the Bcl-x(L) variant and subjected to model apoptotic insults including Sindbis virus (SV) infection, gradual serum withdrawal, and serum deprivation. When cells were engineered to overexpress Bcl-x(L)Delta, cell death due to the SV was inhibited, and Bcl-x(L)Delta provided comparable protection to the wild-type Bcl-x(L) even though expression levels were much lower for the mutant. Furthermore, the cells expressing Bcl-x(L)Delta continued to proliferate following infection while CHO-bcl-x(L) ceased proliferation immediately following infection. As a result, total production of a heterologous protein encoded on the SV was highest in cell lines expressing Bcl-x(L)Delta. Cells expressing the variant Bcl-x(L) also continued to proliferate and showed increased viable cell numbers following gradual serum withdrawal. In contrast, wild-type Bcl-x(L) expressing CHO cells were found to arrest growth but maintain viability following serum withdrawal. Interestingly, CHO cells expressing Bcl-x(L)Delta were also able to recover and return to rapid growth rates much faster than either the wild-type CHO-bcl-x(L) or CHO following the replenishment of fresh complete medium containing 10% FBS. Confocal imaging of yellow fluorescent protein (YFP) fused to the N terminus of Bcl-x(L) and Bcl-x(L)Delta indicated dense aggregates of the Bcl-x(L)Delta while the wild-type protein was distributed throughout the cell in a manner resembling transmembrane localization. As an alternative to complete removal of the loop domain, Bcl-x(L) variants were created in which aspartate residues containing potential caspase recognition sites within the loop domain of Bcl-x(L) were removed. Cell populations expressing various Bcl-x(L)-Asp mutants were exposed to an apoptotic spent medium stimulus, and the cells expressing these Bcl-x(L) variants provided increased viabilities as compared to cells containing wild-type Bcl-x(L) protein. These studies indicate that modification of anti-apoptotic genes can affect multiple cellular properties including response to apoptotic stimuli and cell growth. This knowledge can be valuable in the design of improved apoptosis inhibitors for biotechnology applications.
Figueroa
Bruno
B
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 North Charles Street, 221 Maryland Hall, Baltimore, MD 21218-2694, USA.
Sauerwald
T M
TM
Oyler
G A
GA
Hardwick
J Marie
JM
Betenbaugh
Michael J
MJ
D018354:Alphavirus Infections / Q000503:physiopathology*; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012845:Sindbis Virus; D019311:Staurosporine / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014018:Tissue Distribution; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D016895:Culture Media, Serum-Free; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051020:bcl-X Protein; D019311:Staurosporine