A comparison of the properties of a Bcl-x_L 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_LΔ (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_LΔ, cell death due to the SV was inhibited, and Bcl-x_LΔ 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_LΔ 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_LΔ. 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_LΔ 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 _LΔ indicated dense aggregates of the Bcl-X_LΔ 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.