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Romano Danesi - One of the best experts on this subject based on the ideXlab platform.
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suicide gene therapy with the yeast fusion gene Cytosine Deaminase uracil phosphoribosyltransferase is not enough for pancreatic cancer
Pancreas, 2007Co-Authors: Paola Fogar, Eliana Greco, Carlofederico Zambon, Elisa Fadi, Alessandra Falda, Alessia Stranges, Francesca Vannozzi, Filippo Navaglia, Daniela Basso, Romano DanesiAbstract:Objectives:Suicide gene therapy with FCY1 gene, encoding Cytosine Deaminase (CD), together with FUR1, encoding uracil phosphoribosyltransferase (UPRT), has been proposed for pancreatic cancer therapy in vivo. We ascertained whether gene therapy with FCY1-FUR1 is effective in killing pancreatic cance
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Suicide gene therapy with the yeast fusion gene Cytosine Deaminase/uracil phosphoribosyltransferase is not enough for pancreatic cancer.
Pancreas, 2007Co-Authors: Paola Fogar, Eliana Greco, Carlofederico Zambon, Elisa Fadi, Alessandra Falda, Alessia Stranges, Francesca Vannozzi, Filippo Navaglia, Daniela Basso, Romano DanesiAbstract:Objectives:Suicide gene therapy with FCY1 gene, encoding Cytosine Deaminase (CD), together with FUR1, encoding uracil phosphoribosyltransferase (UPRT), has been proposed for pancreatic cancer therapy in vivo. We ascertained whether gene therapy with FCY1-FUR1 is effective in killing pancreatic cance
Barry L. Stoddard - One of the best experts on this subject based on the ideXlab platform.
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Random mutagenesis and selection of Escherichia coli Cytosine Deaminase for cancer gene therapy
Protein engineering design & selection : PEDS, 2004Co-Authors: Sheri D. Mahan, Greg C. Ireton, Catherine M. Knoeber, Barry L. Stoddard, Margaret E. BlackAbstract:Cytosine Deaminase (CD) is currently being used as a suicide gene for cancer gene therapy. The premise of this therapy is the preferential deamination of 5-fluoroCytosine (5FC) to 5-fluorouracil by cancer cells expressing Cytosine Deaminase. However, a lack of efficient gene transfer to tumors combined with inefficient 5FC turnover currently limits the clinical applications of this gene therapy approach. We have used random mutagenesis to create novel bacterial Cytosine Deaminases that demonstrate an increased preference for 5FC over Cytosine. Among the 15 mutants isolated, one conferred sensitivity to Escherichia coli in a negative selection system at a concentration of 5FC that was 10-fold lower than a sublethal dose for wild-type CD. Evaluation of individual substitutions found in this double mutant (Q102R, D314G) demonstrated that the substitution at residue D314 was solely responsible for the observed increase in sensitivity to 5FC. Additional mutagenesis at D314 resulted in the identification of two more substitutions with the ability to confer enhanced 5FC sensitivity to E.coli. Structure determinations of the three CD variants in the presence and absence of a transition state 5FC analogue provide insights to the determinants of substrate binding specificity at the 5' position of the pyrimidine ring. CD mutant D314A is a promising candidate for further gene therapy studies.
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Alanine-scanning mutagenesis reveals a Cytosine Deaminase mutant with altered substrate preference.
Biochemistry, 2004Co-Authors: Sheri D. Mahan, Greg C. Ireton, Barry L. Stoddard, Margaret E. BlackAbstract:Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes Cytosine Deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluoroCytosine (5-FC). Cytosine Deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial Cytosine Deaminase. The crystal structure of bacterial Cytosine Deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon Cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating Cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in Cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over Cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.
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Microseed matrix screening to improve crystals of yeast Cytosine Deaminase.
Acta crystallographica. Section D Biological crystallography, 2004Co-Authors: Gregory C. Ireton, Barry L. StoddardAbstract:A crystallization strategy termed 'microseed matrix screening' is described where the optimal conditions for nucleation versus extended lattice growth are not compatible. This method is an extension of conventional seeding techniques in which microseeds from the nucleation step are systematically seeded into new conditions where all components of the drop are allowed to vary to screen for subsequent growth of well ordered specimens. The structure of a crystal form of yeast Cytosine Deaminase produced by streak-seeding using a single condition for both nucleation and growth is compared with the structure of a related crystal form produced by separating nucleation and growth conditions. The resulting structural comparison demonstrates that differential chelation patterns of cations by acidic surface residues of proteins within crystal lattice contacts is a critical parameter of crystal nucleation and growth.
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1030. Engineering Cytosine Deaminase for Suicide Gene Therapy
Molecular Therapy, 2004Co-Authors: Margaret E. Black, Sheri D. Mahan, Greg C. Ireton, Catherine M. Knoeber, Barry L. StoddardAbstract:Cytosine Deaminase (CD) is currently being used as a suicide gene for cancer gene therapy. The premise of this therapy is the preferential deamination of 5-fluoroCytosine (5FC) to 5-flurorouracil by cancer cells expressing Cytosine Deaminase. However, a lack of efficient gene transfer to tumors combined with inefficient 5FC turnover currently limits the clinical applications of this gene therapy approach. As a means to address this problem, we have used protein engineering to 1) create novel Cytosine Deaminases that confer increased 5FC sensitivity to cells that express these variants and 2) investigate the role of residues that line the active site pocket with respect to substrate utilization.
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The 1.14 Å Crystal Structure of Yeast Cytosine Deaminase: Evolution of Nucleotide Salvage Enzymes and Implications for Genetic Chemotherapy
Structure (London England : 1993), 2003Co-Authors: Gregory C. Ireton, Margaret E. Black, Barry L. StoddardAbstract:Cytosine Deaminase (CD) catalyzes the deamination of Cytosine and is only present in prokaryotes and fungi, where it is a member of the pyrimidine salvage pathway. The enzyme is of interest both for antimicrobial drug design and gene therapy applications against tumors. The structure of Saccharomyces cerevisiae CD has been determined in the presence and absence of a mechanism-based inhibitor, at 1.14 and 1.43 A resolution, respectively. The enzyme forms an alpha/beta fold similar to bacterial cytidine Deaminase, but with no similarity to the alpha/beta barrel fold used by bacterial Cytosine Deaminase or mammalian adenosine Deaminase. The structures observed for bacterial, fungal, and mammalian nucleic acid Deaminases represent an example of the parallel evolution of two unique protein folds to carry out the same reaction on a diverse array of substrates.
Xandra O. Breakefield - One of the best experts on this subject based on the ideXlab platform.
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Synergistic Anticancer Effects of Ganciclovir/Thymidine Kinase and 5-FluoroCytosine/Cytosine Deaminase Gene Therapies
Journal of the National Cancer Institute, 1998Co-Authors: Manish K. Aghi, Christof M. Kramm, Ting-chao Cho, E. Antonio Chiocca, Xandra O. BreakefieldAbstract:Background: A bacterial enzyme, Escherichia coli Cytosine Deaminase, which converts the prodrug 5-fluoroCytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects. Methods: Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli Cytosine Deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou‐Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied. Results and Conclusions: The presence of transfected Cytosine Deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment. [J Natl Cancer Inst 1998;90: 370‐80]
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synergistic anticancer effects of ganciclovir thymidine kinase and 5 fluoroCytosine Cytosine Deaminase gene therapies
Journal of the National Cancer Institute, 1998Co-Authors: Manish K. Aghi, Christof M. Kramm, Ting-chao Cho, Antonio E Chiocca, Xandra O. BreakefieldAbstract:Background: A bacterial enzyme, Escherichia coli Cytosine Deaminase, which converts the prodrug 5-fluoroCytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects. Methods: Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli Cytosine Deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou‐Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied. Results and Conclusions: The presence of transfected Cytosine Deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment. [J Natl Cancer Inst 1998;90: 370‐80]
Christof M. Kramm - One of the best experts on this subject based on the ideXlab platform.
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mechanisms of thymidine kinase ganciclovir and Cytosine Deaminase 5 fluoroCytosine suicide gene therapy induced cell death in glioma cells
Oncogene, 2005Co-Authors: Ute Fischer, Susanne Frank, Sabine Steffens, Nikolai G. Rainov, Klaus Schulzeosthoff, Christof M. KrammAbstract:Mechanisms of thymidine kinase/ganciclovir and Cytosine Deaminase/ 5-fluoroCytosine suicide gene therapy-induced cell death in glioma cells
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Synergistic Anticancer Effects of Ganciclovir/Thymidine Kinase and 5-FluoroCytosine/Cytosine Deaminase Gene Therapies
Journal of the National Cancer Institute, 1998Co-Authors: Manish K. Aghi, Christof M. Kramm, Ting-chao Cho, E. Antonio Chiocca, Xandra O. BreakefieldAbstract:Background: A bacterial enzyme, Escherichia coli Cytosine Deaminase, which converts the prodrug 5-fluoroCytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects. Methods: Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli Cytosine Deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou‐Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied. Results and Conclusions: The presence of transfected Cytosine Deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment. [J Natl Cancer Inst 1998;90: 370‐80]
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synergistic anticancer effects of ganciclovir thymidine kinase and 5 fluoroCytosine Cytosine Deaminase gene therapies
Journal of the National Cancer Institute, 1998Co-Authors: Manish K. Aghi, Christof M. Kramm, Ting-chao Cho, Antonio E Chiocca, Xandra O. BreakefieldAbstract:Background: A bacterial enzyme, Escherichia coli Cytosine Deaminase, which converts the prodrug 5-fluoroCytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects. Methods: Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli Cytosine Deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou‐Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied. Results and Conclusions: The presence of transfected Cytosine Deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment. [J Natl Cancer Inst 1998;90: 370‐80]
Paola Fogar - One of the best experts on this subject based on the ideXlab platform.
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suicide gene therapy with the yeast fusion gene Cytosine Deaminase uracil phosphoribosyltransferase is not enough for pancreatic cancer
Pancreas, 2007Co-Authors: Paola Fogar, Eliana Greco, Carlofederico Zambon, Elisa Fadi, Alessandra Falda, Alessia Stranges, Francesca Vannozzi, Filippo Navaglia, Daniela Basso, Romano DanesiAbstract:Objectives:Suicide gene therapy with FCY1 gene, encoding Cytosine Deaminase (CD), together with FUR1, encoding uracil phosphoribosyltransferase (UPRT), has been proposed for pancreatic cancer therapy in vivo. We ascertained whether gene therapy with FCY1-FUR1 is effective in killing pancreatic cance
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Suicide gene therapy with the yeast fusion gene Cytosine Deaminase/uracil phosphoribosyltransferase is not enough for pancreatic cancer.
Pancreas, 2007Co-Authors: Paola Fogar, Eliana Greco, Carlofederico Zambon, Elisa Fadi, Alessandra Falda, Alessia Stranges, Francesca Vannozzi, Filippo Navaglia, Daniela Basso, Romano DanesiAbstract:Objectives:Suicide gene therapy with FCY1 gene, encoding Cytosine Deaminase (CD), together with FUR1, encoding uracil phosphoribosyltransferase (UPRT), has been proposed for pancreatic cancer therapy in vivo. We ascertained whether gene therapy with FCY1-FUR1 is effective in killing pancreatic cance
