Biomaterial Database

Redesigned Escherichia coli cytosine deaminase: a new facet of suicide gene therapy. 2015

Asif Raza, and V Kohila, and Siddhartha Sankar Ghosh

Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India.

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.

UI	MeSH Term	Description	Entries
D008175	Lung Neoplasms	Tumors or cancer of the LUNG.	Cancer of Lung,Lung Cancer,Pulmonary Cancer,Pulmonary Neoplasms,Cancer of the Lung,Neoplasms, Lung,Neoplasms, Pulmonary,Cancer, Lung,Cancer, Pulmonary,Cancers, Lung,Cancers, Pulmonary,Lung Cancers,Lung Neoplasm,Neoplasm, Lung,Neoplasm, Pulmonary,Pulmonary Cancers,Pulmonary Neoplasm
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D015316	Genetic Therapy	Techniques and strategies which include the use of coding sequences and other conventional or radical means to transform or modify cells for the purpose of treating or reversing disease conditions.	Gene Therapy,Somatic Gene Therapy,DNA Therapy,Gene Therapy, Somatic,Genetic Therapy, Gametic,Genetic Therapy, Somatic,Therapy, DNA,Therapy, Gene,Therapy, Somatic Gene,Gametic Genetic Therapies,Gametic Genetic Therapy,Genetic Therapies,Genetic Therapies, Gametic,Genetic Therapies, Somatic,Somatic Genetic Therapies,Somatic Genetic Therapy,Therapies, Gametic Genetic,Therapies, Genetic,Therapies, Somatic Genetic,Therapy, Gametic Genetic,Therapy, Genetic,Therapy, Somatic Genetic
D017209	Apoptosis	A regulated cell death mechanism characterized by distinctive morphologic changes in the nucleus and cytoplasm, including the endonucleolytic cleavage of genomic DNA, at regularly spaced, internucleosomal sites, i.e., DNA FRAGMENTATION. It is genetically programmed and serves as a balance to mitosis in regulating the size of animal tissues and in mediating pathologic processes associated with tumor growth.	Apoptosis, Extrinsic Pathway,Apoptosis, Intrinsic Pathway,Caspase-Dependent Apoptosis,Classic Apoptosis,Classical Apoptosis,Programmed Cell Death,Programmed Cell Death, Type I,Apoptoses, Extrinsic Pathway,Apoptoses, Intrinsic Pathway,Apoptosis, Caspase-Dependent,Apoptosis, Classic,Apoptosis, Classical,Caspase Dependent Apoptosis,Cell Death, Programmed,Classic Apoptoses,Extrinsic Pathway Apoptoses,Extrinsic Pathway Apoptosis,Intrinsic Pathway Apoptoses,Intrinsic Pathway Apoptosis
D043525	Cytosine Deaminase	An enzyme which catalyzes the deamination of CYTOSINE resulting in the formation of URACIL. It can also act on 5-methylcytosine to form THYMIDINE.	Cytosine Aminohydrolase,Aminohydrolase, Cytosine,Deaminase, Cytosine
D045744	Cell Line, Tumor	A cell line derived from cultured tumor cells.	Tumor Cell Line,Cell Lines, Tumor,Line, Tumor Cell,Lines, Tumor Cell,Tumor Cell Lines
D019076	Transgenes	Genes that are introduced into an organism using GENE TRANSFER TECHNIQUES.	Recombinant Transgenes,Recombinant Transgene,Transgene,Transgene, Recombinant,Transgenes, Recombinant
D029968	Escherichia coli Proteins	Proteins obtained from ESCHERICHIA COLI.	E coli Proteins