title: Improved cytotoxic effects of Salmonella-producing cytosine deaminase in tumour cells.
abstract: In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate-inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coli codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5-fluorocytosine, a 5-fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures.
authors.affiliation: Centro Andaluz de Biología del Desarrollo, CSIC, Junta de Andalucía, Universidad Pablo de Olavide, Carretera de Utrera, Km. 1, Seville, 41013, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Mesa-Pereira
authors.forename: Carlos
authors.initials: C
authors.lastname: Medina
authors.forename: Eva María
authors.initials: EM
authors.lastname: Camacho
authors.forename: Amando
authors.initials: A
authors.lastname: Flores
authors.forename: Eduardo
authors.initials: E
authors.lastname: Santero
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D001691:Biological Therapy; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D043525:Cytosine Deaminase / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D006801:Humans; D009369:Neoplasms / Q000503:physiopathology / Q000628:therapy*; D012475:Salmonella / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D043525:Cytosine Deaminase