title: 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 field of modern cancer treatment. However, the poor binding affinity of CD towards 5-FC compared to the natural substrate cytosine limits its application for successful 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 F186W mutant and wtCD in a human lung cancer cell line (A549).
  
  
  RESULTS
  A comparative investigation was initiated with cell viability analyses by MTT and trypan blue dye exclusion assays on A549 cells transfected with wtCD and F186W genes. The mode of cell death was confirmed by acridine Orange/ethidium Bromide dual staining. Furthermore, flow cytometric assessments were performed by cell cycle analysis and caspase 3 assay. The experimental results showed a drug dependent decrease in cell viability; interestingly, mutant (F186W) reached IC50 at a much lower concentration of prodrug (5-FC) than wtCD. Cell cycle analysis showed that G1 arrest of a larger population of 5-FC treated F186W transfected cells, in contrast to that of wtCD under similar conditions. The caspase 3 assay revealed progression and execution of apoptosis.
  
  
  CONCLUSIONS
  We report a novel bacterial CD mutant that provided a superior alternate to the wtCD suicide gene. The F186W mutant required a much lower dose of 5-FC to reach its IC50 , thus minimizing the systemic side effects of large doses of 5-FC as required for wtCD.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
authors.forename: Asif
authors.initials: A
authors.lastname: Raza
authors.affiliation: Department of Biotechnology, National Institute of Technology, Warangal, India.
authors.forename: V
authors.initials: V
authors.lastname: Kohila
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India.|Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
authors.forename: Siddhartha Sankar
authors.initials: SS
authors.lastname: Ghosh
mesh_terms: D017209:Apoptosis / Q000235:genetics; D045744:Cell Line, Tumor; D043525:Cytosine Deaminase / Q000235:genetics* / Q000627:therapeutic use; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000627:therapeutic use; D015316:Genetic Therapy; D006801:Humans; D008175:Lung Neoplasms / Q000628:therapy; D009154:Mutation; D019076:Transgenes
chemical_list: D029968:Escherichia coli Proteins; D043525:Cytosine Deaminase; C468827:codA protein, E coli
keywords: cytosine deaminase; mutant; site-directed mutagenesis; suicide gene therapy