Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.4.1 (cytosine deaminase)
747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enzyme activity for cytosine deaminase, which converts cytosine to uracil in bacterial, is usually undetected in higher plants and animals. The enzyme also catalyzes conversion of non-toxic 5-fluorocytosine (5-FC) to 5- fluorouracil (5-FU), a toxic compound for plant growth. The gene encoding cytosine deaminase (codA) from Escherichia coli was fused to cauliflower mosaic virus (CaMV) 35S promoter (P35S), and cloned into a binary vector pLABR101. The resulting plasmid pLABR102 contained two marker genes for plants: a positive marker gene, bialaphos resistance (bar) gene driven by the promoter from nopaline synthase gene (Pnos) and a negative one, P35S-codA. The binary vector pLABR102 was transformed into Arabidopsis thaliana via Agrobacterium-mediated transformation. In transgenic progenies (T3) of the second (T2) generation heterozygous for a single T-DNA insertion, a 3:1 segregation ratio was observed on both bialaphos (resistance to sensitive) and 5-FC (sensitive to unaffected). From T2 plants homozygous for the T-DNA insert, on the other hand, no segregation was detected: all the T3 seedlings were resistant to bialaphos and sensitive to 5-FC. PCR and Northern analyses showed that the 5-FC sensitivity in transgenic descendants was caused by the integration and expression of the chimeric codA gene in the Arabidopsis genome. The results indicated that cytosine deaminase from E. coli is functional and useful for negative selection in Arabidopsis, and that sensitivity to 5-FC as well as the positive bialaphos resistance are dominant traits in Arabidopsis.
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PMID:A conditional negative selection for Arabidopsis expressing a bacterial cytosine deaminase gene. 763 43

The nonmammalian cytosine deaminase (CD) enzyme converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil. Parental cells of a mammary adenocarcinoma (TSA-pc) of BALB/c mice were transfected with the CD gene (TSA-CD), and the ability of 5-FC to hamper their growth was evaluated. A quantity amounting to 0.5 mg of 5-FC/0.3 ml of medium inhibits the proliferation of TSA-CD cells, but not that of TSA-pc, nor that of TSA-pc transfected with neomycin-resistance gene only (TSA-neo). In BALB/c mice, 800 mg 5-FC/kg of body weight injected daily i.p. for 30 days causes total regression of incipient (1-day-old), and established (3- and 7-day-old) TSA-CD tumors, and of 3-day-old experimental lung metastases, but does not impair TSA-pc nor TSA-neo cell growth. Because in CD8+ T lymphocyte- and granulocyte-depleted mice 5-FC no longer impairs TSA-CD growth, immune mechanisms appear to play an important role in this regression. Following, regression, all mice are resistant to subsequent s.c. or i.v. lethal challenges with TSA-pc. The induction of this immune memory is dependent on CD4+ lymphocytes, whereas its effector phase depends on both CD4+ and CD8+ lymphocytes. The memory elicited in tumor-bearing mice by the 5-FC-dependent regression of TSA-CD tumors cures a significant number of mice with 4-day-old TSA-pc metastases, but does not impair the growth of 4-day-old solid s.c. tumors. The reliability of this regression and the subsequent establishment of an efficient immune memory against poorly immunogenic TSA-pc offer the prospect that CD-transduced tumor cells and 5-FC can be used as components of a live antitumor vaccine.
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PMID:5-Fluorocytosine-induced eradication of murine adenocarcinomas engineered to express the cytosine deaminase suicide gene requires host immune competence and leaves an efficient memory. 773 Jun 33

This article reviews uses of metabolic suicide genes in gene therapy. Suicide genes encode novel nonmammalian enzymes that can convert a relatively nontoxic prodrug into a highly toxic agent. Cells genetically transduced to express such genes essentially commit metabolic suicide in the presence of the appropriate prodrug. Three metabolic suicide genes are described: herpes simplex thymidine kinase, Escherichia coli cytosine deaminase and varicella zoster thymidine kinase. Transfer and expression of these genes into mammalian cells is described. Preclinical models of suicide gene therapy of cancer and human immunodeficiency virus are discussed, and several clinical trials employing suicide genes are described.
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PMID:Metabolic suicide genes in gene therapy. 780 80

Several recent reports have demonstrated that anticancer drugs can be generated site-selectively at solid tumors by monoclonal antibody-enzyme conjugates targeted to antigens on tumor cell surfaces. The first step in this drug targeting approach involves the delivery of the enzyme conjugate to a tumor cell population. After the conjugate has localized within the tumor and cleared from non-target tissues, a relatively non-cytotoxic drug precursor (prodrug) is administered. Upon contact with the targeted enzyme, the prodrug is converted into a toxic drug. Several examples are presented to illustrate this targeting strategy. Monoclonal antibody-beta-lactamase conjugates have been developed to activate a panel of anticancer prodrugs that are mechanistically dissimilar. The antitumor activities of the monoclonal antibody-beta-lactamase conjugate/prodrug combinations exceed those obtained by systemic drug administration, and are immunologically specific. In another example involving targeted cytosine deaminase for the generation of 5-fluorouracil, it is shown that as much as 17 times more drug can be delivered within a tumor compared to when 5-fluorouracil is administered alone. The method of using targeted enzymes for prodrug activation can be extended to include prodrugs that release very potent drugs, such as palytoxin, a marine natural product, and to treat cells that have the multidrug resistance phenotype. Some of the requirements for successful therapy with this approach for cancer therapy are discussed.
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PMID:Selective activation of anticancer prodrugs by monoclonal antibody-enzyme conjugates. 788 77

The gene encoding cytosine deaminase (CD) has been expressed in the human colorectal carcinoma cell line WiDr. Metabolism studies confirm that tumor cells expressing CD convert the very nontoxic prodrug 5-fluorocytosine (5FCyt) to 5-fluorouracil (5FUra) and 5FUra metabolites. Tumor xenografts composed of CD-expressing cells can selectively generate tumor levels of > 400 microM 5FUra when the host mouse is dosed with nontoxic levels of 5FCyt. The selective metabolic conversion of 5FCyt to 5FUra in CD-expressing tumor cells results in the inhibition of thymidylate synthase and incorporation of 5FUra into RNA. 5FUra is also liberated into the surrounding environment when CD-expressing tumor cells are treated with 5FCyt. The liberated 5FUra is able to kill neighboring, non-CD-expressing tumor cells in vitro and in vivo. Most importantly, when only 2% of the tumor mass contains CD-expressing cells (98% non-CD-expressing cells), significant regressions in all tumors are observed when the host mouse is dosed with nontoxic levels of 5FCyt.
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PMID:Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. 805 98

Successful expression of the cytosine deaminase (CD) suicide gene in vivo is demonstrated in three weakly immunogenic murine tumor models: the 102 and 205 fibrosarcomas and the 38 adenocarcinoma. Normal mammalian cells do not contain cytosine deaminase, but tumor cells transduced with retroviral vectors containing the CD gene metabolize the relatively nontoxic prodrug 5-fluorocytosine to the highly toxic 5-fluorouracil. In vitro cells expressing the CD gene are killed by 5-fluorocytosine while unmodified cells are not. When injected into syngeneic mice, CD+ tumors can also be eliminated in vivo by systemic treatment with 5-fluorocytosine without significant toxicity to the host. Animals whose CD+ tumors were eliminated with prodrug treatment resist subsequent rechallenge with unmodified wild type tumor. This posttreatment immunity appears to be tumor specific. Applications of the CD system in gene therapy models are discussed.
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PMID:Tumors expressing the cytosine deaminase suicide gene can be eliminated in vivo with 5-fluorocytosine and induce protective immunity to wild type tumor. 813 55

Cytosine deaminase (CDase, EC 3.5.4.1) isolated from Escherichia coli contains a catalytically essential divalent metal ion. Fe2+ was efficiently removed from the enzyme with o-phenanthroline to yield an apoenzyme with less than 5% of the catalytic activity of native enzyme. The time courses for inactivation and for removal of Fe2+ from the enzyme by o-phenanthroline were similar. Apoenzyme reconstituted with Fe2+, Mn2+, Co2+, or Zn2+ (M2+CDase) had kcat values of 185, 88, 50, and 32 s-1, respectively. The Km values of these M2+CDases for cytosine were similar (0.22-0.39 mM). Cytosine potently inhibited reconstitution of the apoenzyme with Fe2+. Fe2+CDase was rapidly inactivated by 1 mM H2O2 (t1/2 < 1 s), whereas Mn2+CDase, Co2+CDase, and Zn2+CDase were not inactivated by H2O2. CDase was also inhibited by excess divalent cations. Cu2+ and Zn2+ reversibly inhibited Fe2+CDase activity with inhibition constants of 1.8 and 5.8 microM, respectively. Cu2+ dissociated slowly from the secondary binding on CDase with a rate constant of 2 x 10(-3) s-1.
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PMID:Cytosine deaminase. The roles of divalent metal ions in catalysis. 822 44

The selective delivery of anticancer drugs to tumors vs normal tissue using targeted antibody-enzyme conjugates for prodrug activation is limited by the amount of drug generated by blood-borne enzyme. Clearance of non-tumor-associated conjugate would increase the tumor/blood conjugate ratio, and enable larger amounts of prodrugs to be administered. A method for clearing the monoclonal antibody (mAb) conjugate L6-cytosine deaminase (L6-CD) was established by using an antibody raised against CD. The mAb 102-26 was obtained by immunizing BALB/C mice with CD conjugated to keyhole limpet hemocyanin. 102-26 was able to precipitate purified CD from solution as assessed by radioimmune precipitation and recognized CD in Western blot analyses. Similar studies were used to establish that 102-26 also recognized CD when conjugated to the L6 and 1F5 mAbs. Selective removal of L6-CD from the circulation of nude mice bearing H2981 human lung adenocarcinoma (L6-antigen positive) was achieved by injecting 102-26 24 h after L6-CD administration. High T/B ratios were obtained by clearance of a L6-CD (38:1 compared to 1.3:1 without clearance).
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PMID:Application of monoclonal antibodies against cytosine deaminase for the in vivo clearance of a cytosine deaminase immunoconjugate. 827 19

A human colorectal carcinoma cell line, WiDr, was genetically engineered to express the nonmammalian enzyme, cytosine deaminase (CD). Expression of CD in WiDr cells (WiDr/CD) did not alter the growth rate of these cells when grown in vitro or as solid tumor xenografts in nude mice. However, expression of CD did increase the sensitivity of these cells to the nontoxic prodrug, 5-fluorocytosine (FCyt), decreasing the 50% inhibitory concentration for FCyt from 26,000 microM in parental WiDr cells to 27 microM in WiDr/CD cells. The increase in sensitivity to FCyt in WiDr/CD cells was the result of the CD-mediated conversion of FCyt to 5-fluorouracil (FUra) and subsequent FUra anabolites. The half-life of the prodrug, FCyt, was determined to be approximately 40 min in nude mice. A single i.p. injection of 500 mg FCyt/kg body weight resulted in a transient FCyt plasma level of approximately 4000 microM while osmotic minipumps or constant tail vein infusions of FCyt achieved continual FCyt plasma levels of 5 microM and 50 microM, respectively, with no overt signs of toxicity. Significant antitumor effects were observed in nude mice bearing tumors derived from WiDr/CD cells when these animals were given 500 mg FCyt/kg i.p. for 10 consecutive days. These antitumor effects were demonstrated by decreases in tumor growth rate, tumor size, tumor weight, and thymidine incorporation into tumor DNA. This antitumor effect was significant but less profound if FCyt was administered by constant tail vein infusion. WiDr and WiDr/CD cells were very sensitive to FUra in vitro (50% inhibitory concentration approximately 5 microM). However, no significant antitumor effects were observed in nude mice bearing tumors derived from either WiDr or WiDr/CD cells when these animals were treated with various doses of FUra. Taken collectively, these data indicate that nontoxic plasma levels of FCyt can be attained which can produce profound antitumor effects on tumors engineered to express CD and that these antitumor effects are significantly better than those that can be achieved using FUra. These positive data support the continued development of a gene therapy approach to colorectal carcinoma involving the selective expression of CD in colorectal tumors with subsequent administration of FCyt.
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PMID:In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. 840 37

We have developed a new approach involving gene therapy for the treatment of primary and metastatic tumors in the liver. As a first step toward the development of this gene therapy treatment for metastatic colorectal carcinoma, the Escherichia coli gene that encodes cytosine deaminase (CD) (EC 3.5.4.1) has been cloned. By using positive genetic selection, a plasmid carrying a 10.8-kilobase BamHI/EcoRI DNA insert was isolated that had CD enzymatic activity. Genetic screening, followed by enzymatic assays, identified a 3-kilobase DNA fragment that retained CD activity. Deamination of cytosine and 5-fluorocytosine (5-FC) by cloned CD was demonstrated. DNA and protein sequencing identified an open reading frame of 427 amino acids that encodes CD. To demonstrate that expression of CD in eukaryotic cells allows metabolism of the nontoxic prodrug 5-FC to the toxic metabolite 5-fluorouracil, CD was cloned into a eukaryotic expression vector and transfected into a human colorectal carcinoma cell line. Growth inhibition studies showed a shift in the IC50 for 5-FC from 17,000 microM in the parental cell line to 30 microM in cells expressing CD.
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PMID:A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase. 845 Aug 32


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