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)

We have been developing an enzyme/prodrug gene therapy approach for the treatment of primary and metastatic tumors in the liver. This system uses the cytosine deaminase/5-fluorocytosine (CD/5-FCyt) enzyme/prodrug combination. Another system that has received considerable attention is the herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) enzyme/prodrug combination. The purpose of the present study was to compare these two enzyme/prodrug systems. The human colorectal tumor cell line, WiDr, was genetically modified to express either the CD gene (WiDr/CD) or the HSV-TK gene (WiDr/TK). The IC50 (concentration of drug producing 50% inhibition of cell growth) for GCV was approximately 3.4 microM in WiDr/TK cells, while the IC50 for 5-FCyt was approximately 27 microM in WiDr/CD cells. In vivo antitumor studies were conducted using high but nontoxic levels of GCV (50 mg/kg/day) or 5-FCyt (500 mg/kg/day). When tumor xenografts were composed of 100% of cells expressing either HSV-TK or CD, 100% tumor-free animals were observed after GCV or 5-FCyt treatment, respectively. However, when only 10% of the tumor cells expressed HSV-TK, no antitumor effect by GCV treatment could be observed. In contrast, when tumors were composed of 4% of the cells expressing CD, 60% of the animals were tumor-free after 5-FCyt treatment. Transmission electron microscopy of the WiDr solid tumors revealed the presence of desmosomes but no gap junctions.
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PMID:Enzyme/prodrug gene therapy: comparison of cytosine deaminase/5-fluorocytosine versus thymidine kinase/ganciclovir enzyme/prodrug systems in a human colorectal carcinoma cell line. 758 11

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

Transduction of malignant cells with toxin genes provides a novel means to promote tumor cell destruction. The efficacy of a toxin gene is dependent on the cell type targeted, the quantity of exogenous protein synthesized, and the mechanisms of growth inhibition and bystander killing. To develop gene therapy for targeting metastatic lung adenocarcinoma, the toxic activity of herpes simplex virus type 1-thymidine kinase, Escherichia coli cytosine deaminase, and human deoxycytidine kinase were investigated in metastatic human lung adenocarcinoma cell lines H1437 and H2122. Cells were transduced stably with retroviral vectors containing the toxin gene cDNA under the control of either a strong [cytomegalovirus (CMV) immediate early promotor and enhancer] or an intermediate strength (Moloney murine leukemia virus long terminal repeat) promotor. A comparison of toxin gene efficacy was based on the level of specific enzyme activity, the concentration of prodrug required to inhibit cell growth by 50%, and the magnitude of the bystander effect. In lung adenocarcinoma cell lines, cytosine deaminase, driven by the CMV promoter, was superior to thymidine kinase and deoxycytidine kinase in its ability to achieve high levels of specific enzyme activity, to induce growth inhibition, and to affect neighboring cell growth. Therefore, cytosine deaminase expressed from the CMV promotor seems to be the most promising toxin gene for human lung adenocarcinoma gene therapy.
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PMID:Comparison of the effects of three different toxin genes and their levels of expression on cell growth and bystander effect in lung adenocarcinoma. 864 Aug 20

To target expression of toxic genes to Epstein-Barr virus (EBV)-associated tumor cells, we have developed an EBV-driven enzyme prodrug system (EDEPS) that takes advantage of the trans-activating properties of EBNA1, a latent protein expressed in all EBV-containing cells, to direct expression of cytosine deaminase (CD) at high levels in those cells only. Plasmids were constructed in which the CD gene or a luciferase reporter gene were cloned downstream of the herpes simplex virus thymidine kinase (tk) promoter and the family of repeats (FR) sequence from the oriP region of EBV. Analysis of luciferase activity after transient transfection into a panel of EBV-negative or -positive human cell lines showed that the presence of the FR element enhanced transcription from the tk promoter in all EBV-positive cell lines, whereas transcription from tk was repressed in all EBV-negative cell lines, including B, T, and fibroblast cell lines. In clonogenicity assays following transfection with the CD vector, the presence of 5-fluorocytosine (5-FC) in the culture medium completely abolished cell growth in EBV-positive cell lines, but did not affect the growth of EBV-negative cell lines. This vector system should have wide applicability in that it allows targeted expression of any gene of interest to tumors that carry EBV, irrespective of the role EBV plays in their pathogenesis.
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PMID:Use of Epstein-Barr virus nuclear antigen-1 in targeted therapy of EBV-associated neoplasia. 884 90

Epstein-Barr virus (EBV)-positive lymphomas are frequent among immunosuppressed patients. We have examined the feasibility of killing EBV-immortalized B lymphocytes by gene transfer involving the use of "suicide" genes whose expression in target cells renders them susceptible to killing by a prodrug. We examined two gene/prodrug pairs: the Escherichia coli cytosine deaminase (CD) gene with the prodrug 5-fluorocytosine (5-FC), and the herpes simplex virus thymidine kinase (HSV-TK) gene with the prodrug ganciclovir. Retroviral vectors and drug selection were used to obtain CD or HSV-TK expression in cells. Both the CD/5-FC and the HSV-TK/ganciclovir combinations yielded substantial killing of EBV-immortalized B lymphocytes in vitro, although the CD/5-FC regimen had a significantly greater therapeutic margin than the HSV-TK/ganciclovir combination. The CD/5-FC pair, but not the HSV-TK/ganciclovir pair, was shown to have a "bystander killing effect" in vitro. When only 30% of the cells expressed the suicide gene, scid mouse tumors regressed in both the CD/5-FC regimen and the HSV-TK/ganciclovir regimen, documenting an in vivo bystander effect with both regimens. However, a greater percentage of tumors completely regressed with the CD/5-FC regimen. Overall, the sum of our data indicates that the CD/5-FC combination is the more promising regimen for treatment of EBV-associated lymphomas in vivo.
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PMID:Killing Epstein-Barr virus-positive B lymphocytes by gene therapy: comparing the efficacy of cytosine deaminase and herpes simplex virus thymidine kinase. 895 14

CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin ] is a prodrug that is converted to the active metabolite SN-38 by carboxylesterases. In its active form, the drug inhibits topoisomerase I, causes DNA damage, and induces apoptosis. Data in this study show metabolism of CPT-11 to SN-38 (7-ethyl-10-hydroxycamptothecin) by a rabbit liver carboxylesterase in vitro and growth-inhibitory activity of the products of the reaction. Additionally, stable expression of the cDNA encoding this protein in Rh30 human rhabdomyosarcoma cells increased the sensitivity of the cells to CPT-11 8.1-fold. We propose that this prodrug/enzyme combination can be exploited therapeutically in a manner analogous to approaches currently under investigation with the combinations of ganciclovir/herpes simplex virus thymidine kinase and 5-fluorocytosine/cytosine deaminase.
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PMID:Overexpression of a rabbit liver carboxylesterase sensitizes human tumor cells to CPT-11. 942 50

Tumor cells that express a fusion gene comprised of Escherichia coli cytosine deaminase (CD) and herpes simplex virus type 1 thymidine kinase (TK) sequences exhibit activation of and subsequent killing by the normally innocuous prodrugs 5-fluorocytosine and ganciclovir (Rogulski et al., Hum. Gene Ther., 8: 73-85, 1997). To target localized expression of this therapeutic gene, we have constructed a recombinant adenovirus containing the CD-TK fusion gene under the control of a human inducible heat shock protein 70 promotional sequence. Strong expression of the fusion gene product was induced by heating at 41 degrees C for 1 h. Expression levels obtained were dependent on the multiplicity of infection used and the incubation time after heat shock. Heat-induced expression of the CD-TK protein significantly reduced the survival of PC-3 cells in the presence of both 5-fluorocytosine and ganciclovir. These studies represent a novel form of gene therapy for the transduction and regulation of a double suicide gene in tumor cells and may provide a unique application for hyperthermia in cancer therapy.
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PMID:Adenoviral-mediated transfer of a heat-inducible double suicide gene into prostate carcinoma cells. 953 29

Suicide genes such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) encode products that convert nontoxic substances (prodrugs) into toxic metabolites. Suicide gene transfer is currently being used in cancer therapy or can be used as a safety modality. To analyze the reliability of suicide genes as a safety modality for a vaccination study with viable cytokine/B7 gene-modified tumor cells, the individual and combined efficacy of the two suicide genes was compared for in vitro and in vivo cell killing of a murine mammary adenocarcinoma cell line (TS/A). To adapt the system to an in vivo gene delivery situation, bulk cultures cotransfected with the CD and TK gene were used instead of selected clones. In vitro, both CD and TK conferred sensitivity to the respective prodrug but the combined cytotoxic effects of both gene products were always superior. For in vivo analysis BALB/c mice were injected subcutaneously with CD- and TK-modified TS/A cells, treated with prodrugs, and tumor size was evaluated for a period of 100 days. In the in vivo situation the combination of both enzyme/prodrug systems was again most effective. The highest single concentration of 5-FC (500 mg/kg) or GCV (100 mg/kg) was not able to fully protect the animals from developing tumors, whereas a combination of 5-FC (250 mg/kg) and GCV (50 mg/kg) resulted in complete tumor eradication. In nude mice treated in the same way, most CD/TK tumors could not be eliminated. Furthermore, BALB/c mice cured of TS/A-CD/TK tumors developed a systemic tumor immunity against challenge with parental TS/A cells. These findings indicate that reliable tumor elimination by the suicide genes depends on T cells. The cooperative effect of both suicide genes was confirmed in vitro with the human renal cell carcinoma line RCC26. We conclude that TK and CD together, but neither gene alone, act as a safety mechanism for the elimination of tumor cells in a reliable fashion and suggest that a rapid and quantitative antigen release by effective TK- and CD-mediated tumor destruction is necessary for T cell immunity to develop.
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PMID:Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo. 958 8

Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer. Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently. The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively. F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells. Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses. CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively. The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr. Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells. These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E. coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E. coli cytosine deaminase. These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.
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PMID:Metabolism and metabolic actions of 6-methylpurine and 2-fluoroadenine in human cells. 963 4

In vitro experiments from our laboratory and others have suggested that herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) gene therapy depends on gap junctional intercellular communication (GJIC) to produce a strong bystander effect. Furthermore, we have shown that cells transduced with HSV-TK can be protected from GCV-mediated toxicity by GJIC with bystander cells. We wished to determine whether GJIC affected either the bystander or protective effect of the cytosine deaminase (CD)/5-flucytosine (5-FC) gene therapy approach, in which CD converts 5-FC to 5-fluorouracil (5-FU). To test this, we designed a coculture system using communication-competent WB rat hepatocytes and a noncommunicating subclone (aB1), which were transduced with CD and with antibiotic resistance genes so that we could independently determine the survival of the CD-containing or bystander cells. We found that, compared to the HSV-TK/GCV strategy, bystander killing resulting from treatment with CD/5-FC does not depend on GJIC. However, our most striking finding was that both communication-competent and -incompetent CD-transduced cells were preferentially killed, by a factor of up to 500, compared to bystander cells. The lesser dependence of the CD/5-FC system on GJIC, combined with the finding that most cancer cells lack the capacity for GJIC, suggest that the CD/5-FC system may be superior to the HSV-TK/GCV approach for gene therapy. However, the premature death of the CD-transduced 5-FU "factory" suggests that other strategies may be necessary to produce a sufficient quantity of 5-FU for a duration long enough to produce permanent tumor regression.
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PMID:Preferential cytotoxicity of cells transduced with cytosine deaminase compared to bystander cells after treatment with 5-flucytosine. 963 83


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