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)

To improve the delivery of so-called suicide genes into tumors, recombinant retroviruses were constructed by inserting the herpes virus type 1 (HSV-1) thymidine kinase (tk), the E. coli cytosine deaminase (cd) and polynucleoside phosphorylase (pnp), or the jellyfish gene for the green fluorescent protein (gfp) into a foamy virus (FV)-derived replication-competent vector (pFOV-7). Expression and stability of the inserted foreign gene was analyzed by immunoblot and polymerase chain reaction (PCR). The functionality of the suicide genes was determined by a metabolic assay on virus vector infected cells and treatment with the respective prodrugs. In terms of vector stability and effectiveness of specific cell killing a virus transducing the pnp gene (FOV-7/pnp) was superior to those using the other two suicide genes. FOV-7/pnp is a candidate virus for suicide gene delivery into solid tumors.
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PMID:Foamy virus vectors for suicide gene therapy. 942 52

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

The parental cells of the TSA murine mammary adenocarcinoma (TSA-pc) were transfected with both the interferon-gamma (IFN-y) gene and the cytosine deaminase (CD) suicide gene to obtain a therapeutic vaccine active against TSA-pc lung metastases. Even in the absence of treatment with the prodrug 5-fluorocytosine (5-FC), the local growth of double transfectants (CD-y clones) was inhibited by a marked recruitment of granulocytes and macrophages. In mice harboring TSA-pc micrometastases, therapeutic vaccination with either IFN-gamma or CD single transfectants reduced the number of lung nodules, whereas CD-gamma double transfectants abrogated metastasis growth in up to 80% of mice. Treatment of mice with 5-FC did not alter the curative efficacy of CD-gamma double-transfectant cells. By contrast, in mice vaccinated with CD single-transfectant cells, 5-FC treatment caused a significant loss of their curative activity. Host T cells played an active role in the cure of lung metastases, because vaccination of nude mice with CD-gamma cells was uneffective.
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PMID:The immune response elicited by mammary adenocarcinoma cells transduced with interferon-gamma and cytosine deaminase genes cures lung metastases by parental cells. 947 81

Cytosine deaminase is an enzyme which has been investigated for cancer chemotherapy as a result of its ability to convert the relatively nontoxic prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil. To facilitate investigations of the utility of cytosine deaminase for cancer chemotherapy, we have cloned and expressed the enzyme from Saccharomyces cerevisiae. The DNA sequence translates into a protein of 158 amino acids in length, with a predicted molecular weight of 17,563 kilodaltons. Alignment of the cytosine deaminase protein sequence from yeast with a variety of proteins defines a novel sequence motif of cytosine or cytidine binding enzymes. Recombinant expression cassettes encoding cytosine deaminase were transfected into monkey kidney COS cells, which lack endogenous cytosine deaminase, to test for production of a functional protein. Cell extracts from these transfectants contained detectable levels of enzyme activity capable of converting 5-fluorocytosine to 5-fluorouracil. Cytosine deaminase was expressed in yeast from a cDNA cassette under the control of an inducible promoter, increasing expression 250- to 300-fold relative to wild-type strains. A purification protocol has been developed which permits recovery of 60% of cytosine deaminase in active form from induced cell lysates after two purification steps. This protocol will be useful for isolating large quantities of pure enzyme which are required for the preclinical evaluation of monoclonal antibody-cytosine deaminase conjugates in combination with 5-fluorocytosine.
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PMID:Cloning, overexpression, and purification of cytosine deaminase from Saccharomyces cerevisiae. 951 58

To explore the antitumor mechanism of bacterial cytosine deaminase plus 5-fluorocytosine (CD/5-FCyt) in combination with interferons (IFNs), glioma cells were transduced with recombinant retroviruses expressing CD. The transduced glioma cells become sensitive to the nontoxic prodrug 5-FCyt. Apoptosis, DNA damage, bystander effect, and inhibition of thymidylate synthase (TS) and DNA synthesis are associated with CD/5-FCyt-mediated glioma cell killing. Furthermore, IFNs enhance this effect by increasing DNA damage and further inhibiting TS activity. The bystander effect is mediated by the release of cytotoxic metabolites of 5-FCyt into the extracellular milieu triggering apoptosis and DNA damage. Our data indicate that the use of CD/5-FCyt in combination with IFNs may provide a more effective approach for the treatment of brain tumors.
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PMID:5-Fluorocytosine-mediated apoptosis and DNA damage in glioma cells engineered to express cytosine deaminase and their enhancement with interferon. 952

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

Direct administration of an adenoviral vector expressing the cytosine deaminase gene (AdCMV.CD) to tumors of colon carcinoma cells, with concomitant systemic administration of 5-fluorocytosine (5FC), results in local production of 5-fluorouracil (5FU) and suppression of tumor growth. Based on the demonstration that in vivo adenovirus-mediated gene transfer to intrahepatic tumors is relatively inefficient compared with in vivo gene transfer to hepatocytes, we developed a 'regional' prodrug strategy using in vivo Ad-mediated CD gene transfer to normal liver, permitting hepatocytes to convert 5FC into 5FU to treat local metastasis effectively in a 'trans' fashion. To show that hepatocytes can generate and export sufficient 5FU to achieve this goal, primary rat hepatocytes were exposed to AdCMV.CD and 5FC. Evaluation of the supernatants by spectrophotometry and by HPLC demonstrated significant conversion of 5FC into 5FU. When supernatants of hepatocytes exposed to AdCMV.CD and 5FC were transferred to cultures of CT26 mouse colon carcinoma cells, the CT26 viability was reduced by 80%. To show that this regional AdCMV.CD/5FC prodrug strategy can suppress tumor growth in vivo, a model of metastatic colon carcinoma was established by injecting CT26 cells into the left lobe of the liver of syngeneic Balb/c mice. The next day, AdCMV.CD was transferred to hepatocytes by intravenous administration, and 5FC treatment was started the following day. Evaluation of tumor growth after 15 days showed marked suppression of tumor growth in AdCMV.CD- and 5FC- treated animals compared to control groups (P < 0.007). We conclude that primary hepatocytes are capable of converting 5FC into 5FU and exporting sufficient amounts of 5FU to the local milieu to suppress the growth of liver metastases of colon carcinoma cells.
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PMID:Regional 'pro-drug' gene therapy: intravenous administration of an adenoviral vector expressing the E. coli cytosine deaminase gene and systemic administration of 5-fluorocytosine suppresses growth of hepatic metastasis of colon carcinoma. 961 75

B cell lymphomas in immunocompromised patients frequently contain the Epstein-Barr virus (EBV) genome (MacMahon et al, 1991), suggesting that gene therapy strategies that target EBV-positive cells for destruction might be useful for the therapy of such tumors. We have previously shown that stable expression of the cytosine deaminase (CD) gene in EBV-positive lymphoblastoid cell lines induces cell killing in the presence of the prodrug 5-fluorocytosine, with a substantial bystander killing effect (Rogers et al., 1996). To promote specific killing of EBV-positive tumor cells, we have constructed two different EBV-based vectors containing the cytosine deaminase gene. The first vector (OriP-CD), which contains the intact EBV oriP enhancer/replication element, replicates as an episome specifically in EBV-positive cells and likewise enhances transcription in an EBV-specific manner. The OriP-CD vector cannot be packaged or spread from cell to cell. The second vector (OriLyt-CD) contains the EBV lytic origin of replication (oriLyt), the EBV packaging sequences (located in the viral termini), the oriP enhancer element (but not the complete replication origin), and the EBV BZLF1 gene (which induces expression of the EBV proteins required for replication of oriLyt). The OriLyt-CD vector is replicated through the oriLyt origin specifically in EBV-positive cells and packaged as an EBV pseudovirion. The packaged oriLyt-CD virion can subsequently infect cells containing the EBV receptor, CD21, and initiate another round of replication in EBV-positive cells. Here we demonstrate that each of these two different EBV-based gene therapy strategies induces specific killing of EBV-positive B cells in vitro (in the presence of 5-FC). The advantages and disadvantages of each strategy are discussed.
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PMID:Gene therapy strategies for treating Epstein-Barr virus-associated lymphomas: comparison of two different Epstein-Barr virus-based vectors. 962 50

Transduction of malignant cells with toxin genes provides a novel strategy by which to promote tumor cell destruction. Whereas the capacity of the toxin gene/prodrug combination cytosine deaminase/fluorocytosine to inhibit growth of human metastatic pulmonary adenocarcinoma cell lines in vitro is established, the in vivo efficacy of this binary system has not yet been determined. For the development of toxin gene therapy for the treatment of lung adenocarcinoma metastatic to the pleural space, a reliable, disease-specific model is required. The serosa of the rat small intestine resembles the basal lamina of the pleura and provides the basis for a more convenient model than direct injection of tumor into the pleural space. Adenocarcinoma cells are inoculated into everted denuded rat intestine configured as a sac. Immunocytochemical and histological analyses show rapid cell growth with characteristics that mimic nodular metastatic intrapleural disease. In the context of this model, systemically delivered fluorocytosine significantly inhibits the growth of cytosine deaminase-expressing human lung adenocarcinoma cells. The dosing schedule required 30 days; neither addition of an enzyme inhibitor that increases the half-life of fluorocytosine nor intralumenal drug delivery is effective in shortening (to 15 days) the protocol. We conclude that CD continues to hold promise as a toxin gene for lung adenocarcinoma gene therapy, and that prolonged prodrug administration may be required for maximum efficacy.
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PMID:Toxin gene-mediated growth inhibition of lung adenocarcinoma in an animal model of pleural malignancy. 962 53


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