Redesigned Escherichia coli cytosine deaminase: A new facet of suicide gene therapy

Abstract

Background The Escherichia coli cytosine deaminase (CD)/5-fluorocytosine(5-FC) approach emerges as a potential aid for suicide gene therapy in the fieldof modern cancer treatment. However, the poor binding affinity of CD towards5-FC compared to the natural substrate cytosine limits its application for suc-cessful suicide gene therapy. Redesigning a bacterial mutant CD with site-directed mutagenesis showed higher potency compare to wild-type CD (wtCD)in vitro. In the present study, we conducted a comparative analysis of F186Wmutant and wtCD in a human lung cancer cell line (A549).Methods and Results A comparative investigation was initiated with cellviability analyses by MTT and trypan blue dye exclusion assays on A549 cellstransfected with wtCD and F186W genes. The mode of cell death wasconfirmed by acridine Orange/ethidium Bromide dual staining. Furthermore,flow cytometric assessments were performed by cell cycle analysis and caspase3 assay. The experimental results showed a drug dependent decrease in cellviability; interestingly, mutant (F186W) reached IC50 at a much lower concen-tration of prodrug (5-FC) than wtCD. Cell cycle analysis showed that G1 arrestof a larger population of 5-FC treated F186W transfected cells, in contrast tothat of wtCD under similar conditions. The caspase 3 assay revealed progres-sion and execution of apoptosis.Conclusions We report a novel bacterial CD mutant that provided a superioralternate to the wtCD suicide gene. The F186W mutant required a much lowerdose of 5-FC to reach its IC 50 , thus minimizing the systemic side effects of largedoses of 5-FC as required for wtCD.