A comparison of the properties of a Bcl-x L variant to the wild-type anti-apoptosis inhibitor in mammalian cell cultures

Abstract

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.