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)

Enzymes, the expression products of transferred or native genes, offer unique windows of opportunity for clinical diagnosis and therapy. Although some expression products can be monitored in plasma, nuclear medicine imaging (SPECT and PET) offers the unique ability to selectively measure the intensity and regional/spatial distribution of gene expression both in vivo, in situ. Importantly, the superior sensitivity and moderate spatial resolution of the nuclear techniques also enable in vivo kinetic characterization of enzyme-substrate interaction. Indeed, the non-invasive, whole-body assessment of gene expression can only be achieved through imaging techniques. Given today's technology, nuclear imaging techniques uniquely provide the necessary sensitivity required to evaluate the success of the gene delivery and expression (transcription and translation), and to detect unwanted expression by non-target tissues. Enzymes are a special class of proteinacious gene expression products that selectively bind specific substrates for the purpose of molecular biotransformation rather than for signal transduction. In general, enzymes have received much less attention for imaging than receptors and antibodies, despite the enzymes' high substrate specificity and the potential for kinetic evaluation. Enzymes are attractive targets for diagnostic imaging and radioisotope radiotherapy because they convert multiple molecular copies of the substrate (radiotracer) per molecule of enzyme, thereby greatly increasing the ultimate sensitivity relative to the sensitivity offered by receptors that bind with 1:1 stoichiometry. Not surprisingly, enzymes have been the preferred molecular targets to date for scintigraphic imaging of gene therapy. This overview describes opportunities and advances in the utilization of radiolabelled nucleosides and nucleoside bases for imaging in gene therapy, with emphasis on the exploitation of enzyme systems for scintigraphic imaging of gene expression in gene therapy of cancer. Herpes simplex virus type-1 thymidine kinase and bacterial/fungal cytosine deaminase are discussed within the context of gene therapy issues such as gene vectors for targeting and delivery, the bystander effect, and radionucleoside delivery. The utilization of nucleosides as markers of tissue proliferation is discussed with respect to selected enzyme targets.
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PMID:Enzyme-targeted, nucleoside-based radiopharmaceuticals for scintigraphic monitoring of gene transfer and expression. 1177 56

The genome sequence of Plasmodium falciparum, the causative agent of the most severe form of malaria in humans, rapidly approaches completion, but our ability to genetically manipulate this organism remains limited. Chromosomal integration has only been achieved following the prolonged maintenance of circularised episomal plasmids which selects for single crossover recombinants. It has not been possible to construct genetic deletions via double crossover recombination, presumably due to the low frequency of this event. We have used the Herpes simplex virus thymidine kinase gene and the Escherichia coli cytosine deaminase gene for negative selection of P. falciparum. Parasites were transformed with plasmids expressing the thymidine kinase and cytosine deaminase genes by positive selection for the human dihydrofolate reductase gene. Parasites expressing thymidine kinase are susceptible to the pro-drug ganciclovir while those expressing cytosine deaminase are sensitive to 5-fluorocytosine. Parental parasites were inherently resistant to these drugs. A significant 'bystander effect' was evident in cultures with either ganciclovir or 5-fluorocytosine. Positive and negative selection of the thymidine kinase transformants with both ganciclovir and WR99210 resulted in the selection of parasites containing a genetic deletion of the Pfrh3 gene, the first targeted double crossover deletions in P. falciparum. The use of negative selection for gene disruptions via double crossover recombination will dramatically improve our ability to analyse protein function and opens the possibility of using this strategy for a variety of gene deletion and modification experiments in the analysis of this important infectious agent.
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PMID:Negative selection of Plasmodium falciparum reveals targeted gene deletion by double crossover recombination. 1179 25

We have previously developed a recombinant adenovirus containing a fusion gene of Escherichia coli cytosine deaminase (CD) and herpes simplex virus type 1 thymidine kinase (HSV-1 TK) controlled by a cytomegalovirus (CMV) enhancer-promoter. This replication-incompetent adenovirus effectively transduced the CD-TK gene into human prostate adenocarcinoma DU-145 or PC-3 cells. Interestingly, heat shock at 41 degrees C for 4 hours elevated the level of CD-TK by approximately 5- to 20-fold at a multiplicity of infection (MOI) of 1. Heat-enhanced expression of CD-TK promoted cytotoxicity by 23-, 9-, or 47-fold in the presence of 50 microg/mL ganciclovir (GCV), 500 microg/mL 5-fluorocytosine (5-FC), or 50 microg/mL GCV+500 microg/mL 5-FC, respectively, at an MOI of 1. Moreover, there was an increase in radiosensitivity when adenovirus-infected cells were heated at 41 degrees C for 4 hours followed by irradiation in the presence of the prodrugs. Virus+heat+1 microg/mL GCV treatment increased radiosensitivity by a dose-modifying factor (DMF) of 2.2, whereas virus+heat+10 microg/mL 5-FC exposure resulted in a DMF of 2.3. Radiosensitization was clearly enhanced as a result of combined prodrug exposure (DMF=4.4). Our results suggest that the efficiency in expression of suicide genes from an adenoviral vector used for cytotoxic anticancer therapy could be improved by combining heat treatment with radiation therapy.
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PMID:Gene transfer into human prostate adenocarcinoma cells with an adenoviral vector: Hyperthermia enhances a double suicide gene expression, cytotoxicity and radiotoxicity. 1189 43

Standard chemotherapeutic agents and ionizing radiation destroy dividing cells. Because tumor cells divide more rapidly than normal cells, there is a therapeutic index in which damage to the cancer cells is maximized while keeping the toxicity to the normal host cells acceptable. Suicide gene therapy strives to deliver genes to the cancer cells, which convert nontoxic prodrugs into active chemotherapeutic agents. With this strategy, the systemically administered prodrug is converted to the active chemotherapeutic agent only in cancer cells, thereby allowing a maximal therapeutic effect while limiting systemic toxicity. A literature search was conducted using the MEDLINE database from 1990 to 2001 to identify articles related to suicide gene therapy for cancer. A number of suicide gene systems have been identified, including the herpes simplex virus thymidine kinase gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the E. coli Deo gene. Various vectors, including liposomes, retroviruses, and adenoviruses, have been used to transfer these suicide genes to tumor cells. These strategies have been effective in cell culture experiments, laboratory animals, and some early clinical trials. Advances in tissue- and cell-specific delivery of suicide genes using specific promoters will improve the clinical utility of suicide gene therapy.
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PMID:Current progress in suicide gene therapy for cancer. 1194 67

Herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli cytosine deaminase (CD) are non-mammalian enzymes capable of converting innocuous prodrugs into cytotoxic metabolites. Both enzymes have been utilized independently, as well as together in 'suicide' gene therapy protocols to eliminate tumor cells in vitro and in vivo. We have used a set of replication defective HSV vectors expressing either or both enzymes to compare the efficacies of single and double suicide gene therapies in the 9L gliosarcoma model in vitro and in vivo. In cell culture experiments at high and low multiplicities of infection, combined expression of the two genes by vector TOCD/TK along with exposure to the matching prodrugs (ganciclovir and 5-fluorocytosine) showed increased cytotoxicity compared with exposure to either prodrug alone. However, the two gene combination was inferior to single gene treatments, suggesting that HSVtk and CD are mutually counteractive in the prodrug-dependent killing of glioma cells. In animal experiments, survival was not significantly prolonged by administration of both prodrugs to TOCD/TK-treated animals, while each single gene/prodrug pair resulted in increased survival. These results indicate that single suicide gene systems employing HSVtk or CD may be preferable over combinations of the two.
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PMID:Double suicide gene therapy using a replication defective herpes simplex virus vector reveals reciprocal interference in a malignant glioma model. 1197 34

Clinical gene therapy needs non invasive tools to evaluate the efficiency of gene transfer. This includes the evaluation of infection efficiency as well as the verification of successful gene transfer in terms of gene transcription. These informations can be used for therapy planning, follow up studies in treated tumors and as an indicator of prognosis. Therapy planning is performed by the assessment of gene expression for example using radiolabeled specific substrates to determine the activity of suicide enzymes as the Herpes Simplex Virus thymidine kinase or cytosine deaminase. Furthermore, other in vivo reporter genes as receptors, antigens or transport proteins may be used in bicistronic vector systems for the evaluation of gene transduction and expression. This is done using radiolabeled ligands, antigens or substrates. Follow up studies with magnetic resonance imaging, single photon emission tomography or positron emission tomography may be done to evaluate early or late effects of gene therapy on tumor volume, metabolism or proliferation. Finally, enhancement of radioactive isotope accumulation in tumors by transfer of the appropriate genes may be used for the treatment of malignant tumors.
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PMID:Imaging methods in gene therapy of cancer. 1210 53

Adenovirus-mediated suicide gene therapy may hold promise in the treatment of human cancer. We have developed a novel approach that utilizes a lytic, replication-competent adenovirus (Ad5-CD/TKrep) to deliver a cytosine deaminase/herpes simplex virus-1 thymidine kinase fusion gene to tumors. The cytosine deaminase and herpes simplex virus-1 thymidine kinase suicide genes render malignant cells sensitive to specific pharmacological agents and, importantly, sensitize them to radiation. The Phase I study described here represents the first gene therapy trial in which a replication-competent virus was used to deliver a therapeutic gene to humans. The indication is local recurrence of prostate cancer after definitive radiation therapy. An escalating dose (10(10), 10(11), and 10(12) viral particles) of the Ad5-CD/TKrep virus was injected intraprostatically under transrectal ultrasound guidance into 16 patients in four cohorts. Two days later, patients were given 5-fluorocytosine and ganciclovir prodrug therapy for 1 (cohorts 1-3) or 2 (cohort 4) weeks. There were no dose-limiting toxicities, and the maximum tolerated dose of the Ad5-CD/TKrep vector was not defined. Ninety-four percent of the adverse events observed were mild or moderate (grade 1/2) in nature. Seven of 16 (44%) patients demonstrated a >or=25% decrease in serum prostate-specific antigen, and 3 of 16 (19%) patients demonstrated a >or=50% decrease in serum prostate-specific antigen. Transgene expression and tumor destruction at the injection site were confirmed by sextant needle biopsy of the prostate at 2 weeks. Two patients were negative for adenocarcinoma at 1 year follow-up. Although Ad5-CD/TKrep viral DNA could be detected in blood as far out as day 76, no infectious adenovirus was detected in patient serum or urine. Together, the results demonstrate that intraprostatic administration of the replication-competent Ad5-CD/TKrep virus followed by 2 weeks of 5-fluorocytosine and ganciclovir prodrug therapy can be safely applied to humans and is showing signs of biological activity.
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PMID:Phase I study of replication-competent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer. 1220 48

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.
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PMID:Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers. 1224 48

Molecular imaging is broadly defined as the characterization and measurement of biological processes in living animals, model systems, and humans at the cellular and molecular level using remote imaging detectors. One underlying premise of molecular imaging is that this emerging field is not defined by the imaging technologies that underpin acquisition of the final image per se, but rather is driven by the underlying biological questions. In practice, the choice of imaging modality and probe is usually reduced to choosing between high spatial resolution and high sensitivity to address a given biological system. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) inherently use image-enhancing agents (radiopharmaceuticals) that are synthesized at sufficiently high specific activity to enable use of tracer concentrations of the compound (picomolar to nanomolar) for detecting molecular signals while providing the desired levels of image contrast. The tracer technologies strategically provide high sensitivity for imaging small-capacity molecular systems in vivo (receptors, enzymes, transporters) at a cost of lower spatial resolution than other technologies. We review several significant PET and SPECT advances in imaging receptors (somatostatin receptor subtypes, neurotensin receptor subtypes, alpha(v)beta(3) integrin), enzymes (hexokinase, thymidine kinase), transporters (MDR1 P-glycoprotein, sodium-iodide symporter), and permeation peptides (human immunodeficiency virus type 1 (HIV-1) Tat conjugates), as well as innovative reporter gene constructs (herpes simplex virus 1 thymidine kinase, somatostatin receptor subtype 2, cytosine deaminase) for imaging gene promoter activation and repression, signal transduction pathways, and protein-protein interactions in vivo.
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PMID:Molecular imaging of gene expression and protein function in vivo with PET and SPECT. 1235 50

The rat prostate tumour cell line R3327 AT-1 was transfected with a gene coding for a fusion protein comprised of cytosine deaminase (CD from E. coli) and thymidine kinase (TK from Herpes simplex virus, HSV-1). The resulting AT-1/CDglyTK cell line was sensitive to the prodrug 5-fluorocytosine (IC(50) = 78 microM, 96-h incubation) via CD and to ganciclovir (GCV, IC(50) = 1 microM, 96 h) via TK. Subcutaneous tumours generated from 100% CDglyTK(+) cells responded well to 5-FC therapy (500 mg/kg, i.p., 14 daily treatments, four out of seven animals in remission) and to GCV therapy (30 mg/kg, i.p., 14 daily treatments, five of six animals in remission). However, experiments with mixtures of CDglyTK(+) and CDglyTK(-) cells showed low levels of connexins (intercellular gap junctions) and no bystander effect for nontransfected cells using either 5-FC or GCV therapy. Furthermore, (19)F-NMR spectroscopy showed that incubation of cultured CDglyTK(+) cells with 774 microM 5-FC for 16 h resulted in the following intracellular concentrations: 5-FC = 314 microM, 5-FU = 52 microM, cytotoxic fluoronucleotides = 163 microM; extracellular 5-FU reached only 6.4 microM. Thus, in this model system intracellular trapping of 5-FU (slow export) contributes to the failure of the CD/5-FC bystander effect via an extracellular route.
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PMID:Cytosine deaminase and thymidine kinase gene therapy in a Dunning rat prostate tumour model: absence of bystander effects and characterisation of 5-fluorocytosine metabolism with 19F-NMR spectroscopy. 1242 9


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