Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.4.1 (cytosine deaminase)
 747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
Cancer Res 2002 Sep 01
PMID:Phase I study of replication-competent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer. 1220 48

Tumor cell contamination of clinical grafts is a major concern in autologous hematopoietic stem cell transplantation because these contaminating cells can contribute to relapse. In the present work, we use a suicide gene therapy approach that successfully accomplishes the two main goals of any purging strategy: highly efficient elimination of contaminating tumor cells and preservation of the engraftment capability of the hematopoietic progenitor cells. Human CD34(+) cells spiked with breast cancer cells were infected with an adenoviral vector encoding the cytosine deaminase transgene (Ad-CMV-CD). In vitro, transduction with Ad-CMV-CD followed by exposure to 400 micro M 5-fluorocytosine resulted in complete elimination of clonogenic contaminating tumor cells without affecting the clonogenic potential of the human hematopoietic CD34(+) cells. Transplantation of nonobese diabetic/LtSz-scid/severe combined immunodeficient (NOD/SCID) mice with nonpurged contaminated grafts and purged contaminated grafts, allowed us to test the safety and efficacy of our procedure in two independent purging experiments. Hematopoietic engraftment kinetics as well as the quantity and quality of human engraftment were not affected by the purging therapy. Results showed a significant difference in survival between the nonpurged group (28%) and the purged group (100%; P = 0.012). Moreover, highly sensitive histological and molecular analyses confirmed the absence of tumor cells in the recipients of purged marrow. In contrast, metastatic tumors were detected in animals that received nonpurged grafts. We anticipate that this strategy will result in a safe and efficacious hematopoietic graft product for autologous transplantation for patients with multiple forms of epithelial cancers.
Cancer Res 2002 Sep 01
PMID:Efficient and nontoxic adenoviral purging method for autologous transplantation in breast cancer patients. 1220 55

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.
Planta 2002 Sep
PMID:Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers. 1224 48

With the goal of optimizing adenovirus-mediated suicide gene therapy for prostate cancer, we have developed a method based on the human sodium iodide symporter (hNIS) that allows for noninvasive monitoring of adenoviral vectors and quantification of gene expression magnitude and volume within the prostate. A replication-competent adenovirus (Ad5-yCD/mutTK(SR39)rep-hNIS) coexpressing a therapeutic yeast cytosine deaminase (yCD)/mutant herpes simplex virus thymidine kinase (mutTK(SR39)) fusion gene and the hNIS gene was developed. Ad5-yCD/mutTK(SR39)rep-hNIS and a replication-defective hNIS adenovirus (rAd-CMV-FLhNIS) were injected into contralateral lobes of the dog prostate and hNIS activity was monitored in live animals following administration of Na(99m)TcO(4) using gamma camera scintigraphy. Despite the close proximity of the urinary bladder, (99m)TcO(4)(-) uptake was readily detected in the prostate using viral dose levels (10(10) to 10(12) viral particles) that have been safely administered to humans. Due to its rapid clearance and short physical half-life (6 h), it was possible to obtain daily measurements of (99m)TcO(4)(-) uptake in vivo, allowing for dynamic monitoring of reporter gene expression within the prostate as well as biodistribution throughout the body. High-resolution autoradiography of prostate sections coupled with 3D reconstruction of gene expression demonstrated that the magnitude and volume of gene expression could be quantified with submillimeter resolution. Implementation of the GENIS (gene expression of Na/I symporter) technology in the clinic will facilitate optimization of future human gene therapy trials.
Mol Ther 2003 Sep
PMID:GENIS: gene expression of sodium iodide symporter for noninvasive imaging of gene therapy vectors and quantification of gene expression in vivo. 1294 25

EGP-2, also known as Ep-CAM, is expressed at high levels on the surface of most carcinomas and is therefore considered an attractive target for anticancer strategies. To explore the mechanisms regulating the expression of EGP-2, sequences 3.4 kb upstream of the transcription start site were isolated and assayed for their ability to control the expression of the EGP-2 cDNA, the green fluorescent protein, the luciferase reporter gene and the thymidine kinase and cytosine deaminase suicide genes. Expression of these chimeric constructs as assessed in a range of different cell lines was restricted to cell lines expressing EGP-2. In addition, only cells expressing EGP-2 were sensitive for gancyclovir after being transiently transfected with EGP-2 promoter-driven thymidine kinase. Deletion analyses defined 687 bp upstream as the basic proximal promoter region, which could confer epithelial-specific expression to the GFP reporter gene in vitro. As these EGP-2 sequences can confer promoter activity to reporter and suicide genes in an EGP-2 restricted manner, they may be useful for gene therapy of EGP-2 expressing carcinomas.
Cancer Gene Ther 2004 Sep
PMID:Use of the EGP-2/Ep-CAM promoter for targeted expression of heterologous genes in carcinoma derived cell lines. 1524 30

Class switch recombination (CSR) at the DNA level underlies ability of B lymphocytes to switch from expressing IgM to expressing IgG, IgA, or IgE. The mechanism of CSR is largely unknown, but it is clear that CSR is stimulated by T cell signals and is mediated in part by activation-induced deaminase (AID), an enzyme that is also required for somatic hypermutation of Ig genes. In one current model, AID is proposed to initiate CSR by deaminating cytosines in the unpaired nontemplate strand of DNA displaced from its complementary strand by the "sterile" RNA transcript across the switch region. We have used LM-PCR to analyze single-strand breaks in CH12F3-2, a murine cell line that switches in vitro to IgA expression. In contrast to the above model, we have detected CSR-associated ssDNA breaks in the template strand of the H chain alpha switch region, the strand thought to be complexed with RNA. Most breaks are adjacent to cytosines, consistent with mediation by AID, and occur within the novel consensus sequence C*AG, which occurs much more frequently on the template strand than on the putatively displaced nontemplate strand. These results suggest that AID may target the DNA strand bound to RNA, perhaps resembling APOBEC-3G, a cytosine deaminase related to AID that inhibits HIV replication by mutating viral DNA. Furthermore, the absence of detectable breaks in the nontemplate strand within the DNA segment under study suggests that the two DNA strands are handled differently in the generation or processing of strand breaks.
J Immunol 2004 Sep 01
PMID:Single-stranded DNA breaks adjacent to cytosines occur during Ig gene class switch recombination. 1532 84

We constructed an oncolytic adenoviral vector Ad.HE1HCD3, in which the adenoviral E1A promoter was replaced by a human tyrosinase enhancer (HTE)/promoter. The RGD-4C peptide was inserted into the HI loop of the fiber knob domain to increase the transduction efficiency of this vector for tumor cell lines. We also inserted the prodrug activating cytosine deaminase gene driven by the HTE/promoter into the E3 region of the Ad.HE1HCD3 vector. The in vitro cytotoxic effect of the Ad.HE1HCD3 vector with 5-fluorocytosine (5-FC) was greater than that of a wild-type adenovirus or that of the Ad.HE1HCD3 vector alone in tyrosinase-positive melanoma cell lines at low multiplicity of infection. Intratumoral injection of low doses of the Ad.HE1HCD3 vector into xenotransplanted human melanoma cell lines followed by the intraperitoneal injection of 5-FC led to a greater degree of tumor regression in vivo than did the intratumoral injection of the same dose of the Ad.HE1HCD3 vector alone. This oncolytic vector with a melanoma-specific prodrug activation therapeutic transcription unit and a RGD targeted fiber protein offers a potent therapeutic combination for the gene therapy of melanoma.
Cancer Gene Ther 2006 Sep
PMID:Oncolytic adenoviral vector carrying the cytosine deaminase gene for melanoma gene therapy. 1671 Mar 44

Alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) is a widespread enzyme found in many bacterial species and all currently sequenced eukaryotic organisms. It occupies a key position at the branching point of two metabolic pathways: the tryptophan to quinolinate pathway and the bacterial 2-nitrobenzoic acid degradation pathway. The activity of ACMSD determines whether the metabolites in both pathways are converted to quinolinic acid for NAD biosynthesis or to acetyl-CoA for the citric acid cycle. Here we report the first high-resolution crystal structure of ACMSD from Pseudomonas fluorescens which validates our previous predictions that this enzyme is a member of the metal-dependent amidohydrolase superfamily of the (beta/alpha)(8) TIM barrel fold. The structure of the enzyme in its native form, determined at 1.65 A resolution, reveals the precise spatial arrangement of the active site metal center and identifies a potential substrate-binding pocket. The identity of the native active site metal was determined to be Zn. Also determined was the structure of the enzyme complexed with cobalt at 2.50 A resolution. The hydrogen bonding network around the metal center suggests that Arg51 and His228 may play important roles in catalysis. The metal center configuration of PfACMSD is very similar to that of Zn-dependent adenosine deaminase and Fe-dependent cytosine deaminase, suggesting that ACMSD may share certain similarities in its catalytic mechanism with these enzymes. These data enable us to propose possible catalytic mechanisms for ACMSD which appear to be unprecedented among all currently characterized decarboxylases.
Biochemistry 2006 Sep 05
PMID:Crystal structure of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase: insight into the active site and catalytic mechanism of a novel decarboxylation reaction. 1693 94

In preparation for a Phase I trial, we evaluated the efficacy and toxicity of replication-competent adenovirus-mediated suicide gene therapy in combination with radiation in a preclinical model of pancreatic cancer. Human MiaPaCa-2 and PANC-1 pancreatic adenocarcinoma cells were found to be sensitive to the oncolytic effects of the Ad5-yCD/mutTK(SR39)rep-ADP adenovirus and also to the cytotoxic effects of the yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK(SR39)) genes in vitro. Combining Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy with radiation significantly increased tumor control beyond that of either modality alone. Injection of Ad5-yCD/mutTK(SR39)rep-ADP in the dog pancreas at doses (10(12) virus particle (vp)) to be used in humans resulted in mild pancreatitis but not peritonitis or hepatotoxicity. Following administration of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]-FHBG), a positron-emitting substrate of HSV-1 TK, Ad5-yCD/mutTK(SR39)rep-ADP activity could be monitored non-invasively by positron emission tomography (PET). [(18)F]-FHBG uptake was readily detected in the pancreas but not in other major abdominal organs, indicating that little of the injected adenovirus disseminates to collateral tissues. These results demonstrate that Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy has the potential to augment the effectiveness of pancreatic radiotherapy without resulting in excessive toxicity. Hence they provide the scientific basis for an ongoing Phase I trial in pancreatic cancer.
Mol Ther 2007 Sep
PMID:Replication-competent adenovirus-mediated suicide gene therapy with radiation in a preclinical model of pancreatic cancer. 1755 7

The human APOBEC3G protein is an innate anti-viral factor that can dominantly inhibit the replication of some endogenous and exogenous retroviruses. The prospects of purposefully harnessing such an anti-viral defense are under investigation. Here, long-term co-culture experiments were used to show that porcine endogenous retrovirus (PERV) transmission from pig to human cells is reduced to nearly undetectable levels by expressing human APOBEC3G in virus-producing pig kidney cells. Inhibition occurred by a deamination-independent mechanism, likely after particle production but before the virus could immortalize by integration into human genomic DNA. PERV inhibition did not require the DNA cytosine deaminase activity of APOBEC3G and, correspondingly, APOBEC3G-attributable hypermutations were not detected. In contrast, over-expression of the sole endogenous APOBEC3 protein of pigs failed to interfere significantly with PERV transmission. Together, these data constitute the first proof-of-principle demonstration that APOBEC3 proteins can be used to fortify the innate anti-viral defenses of cells to prevent the zoonotic transmission of an endogenous retrovirus. These studies suggest that human APOBEC3G-transgenic pigs will provide safer, PERV-less xenotransplantation resources and that analogous cross-species APOBEC3-dependent restriction strategies may be useful for thwarting other endogenous as well as exogenous retrovirus infections.
PLoS One 2007 Sep 12
PMID:The restriction of zoonotic PERV transmission by human APOBEC3G. 1784 22


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