Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU), and has been investigated extensively as a potential tool for selective cellular eradication. In this paper, genetic constructs were designed to express the CD enzyme fused to the transmembrane and extracellular domains of the human nerve growth factor receptor (NGFR), thus allowing for positive identification of transduced cells by flow cytometry and positive selection by magnetic bead technology. Constructs were designed to encode a [Gly(4)Ser](2) flexible linker between the nucleic acid coding sequences for the NGFR and CD genes. Retroviral vectors constructed with wild-type CD and NG/CD fusion genes were used to transduce 3T3 fibroblasts and the human T cell line CEM. The function of CD fusion genes was comparable to that of wild-type genes as determined in cytotoxicity assays. By flow cytometry, the NGFR antigen was detectable after expression of the fusion gene derived from either Escherichia coli (NG/CDe) or Saccharomyces cerevisiae (NG/CDs), but the greatest antigen density was observed in cells transduced with the NG/CDs vector. Similarly, superior 5-FC sensitivity was observed with NG/CDs fusion gene in both murine fibroblasts and human T cells. In addition, CEM cells expressing NG/CDs were more efficiently eliminated in vivo. Engineering of cells utilizing the chimeric NG/CD genes provides a new modality in gene therapy allowing positive and negative selection using a single protein-coding sequence.
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PMID:Human nerve growth factor receptor and cytosine deaminase fusion genes. 1286 18

Soft tissue and bone sarcomas of the extremities can be difficult to eradicate, and standard treatment may require limb amputation. New therapies to decrease tumor size could improve the effectiveness of treatment and decrease the frequency of limb amputation. Cytosine deaminase (CD)-based gene therapy has been shown to be effective in decreasing growth of solid tumors when animals with CD-expressing tumor cells are treated with 5 fluorocytosine (5FC), an inert prodrug that is converted to 5-fluorouracil (5FU) by CD. In this investigation, we used a novel CD-containing fusion gene to determine whether CD-based gene therapy affected soft tissue or bone sarcomas. The novel fusion gene (NGFR-CD) encodes for a protein with extracellular and transmembrane domains of human nerve growth factor receptor (NGFR) and cytoplasmic CD. Murine 2472 (2) sarcoma cells were transduced with fusion genes containing either the bacterial (NGFR-(b)CD) or yeast (NGFR-(y)CD) CD gene. 5FC treatment killed NGFR-(b)CD- and NGFR-(y)CD-transduced sarcoma cells in vitro through direct and bystander effects (P < 0.01). In contrast, 5FC treatment of mice with s.c. 2NGFR-(b)CD or 2NGFR-(y)CD tumors affected only 2NGFR-(y)CD tumors. 5FC had no effect on growth of NGFR-(b)CD tumors but caused significant decrease in the size of 2NGFR-(y)CD tumors (51 +/- 60 versus 938 +/- 767 mm(3), treated versus control, P < 0.01). Evaluation of bystander killing in vivo revealed significant tumor killing, with a 5-fold reduction in s.c. tumor volume evident in saline versus 5FC-treated mice when tumors were comprised of 90% 2472 cells and 10% 2NGFR-(y)CD selected for fluorescence-activated cell sorting (P < 0.01). Bone sarcomas were eliminated in 9 of 10 5FC-treated mice, compared with 11.8 +/- 6.0 mm(2) in saline-treated mice (P < 0.002). In addition, 5FC treatment of bone sarcomas caused a significant reduction in cancer-induced bone destruction (P < 0.002) and resulted in a reduction in the number of osteoclasts. Finally, 5FC treatment had no effect on animal weight or survival, whereas doses of 5FU providing equivalent tumor reduction as 5FC resulted in treatment-associated deaths and significant weight loss (P < 0.001).
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PMID:Direct and bystander killing of sarcomas by novel cytosine deaminase fusion gene. 1458 82

Cytosine deaminase (CD) catalyzes the deamination of 5-fluorocytosine (5FC) to produce the highly toxic chemotherapeutic agent 5-fluorouracil (5FU). A unique feature of the CD/5FC enzyme/prodrug system is its ability to kill adjacent cells via bystander killing. Bystander killing of cancer cells can be mediated by non-cancerous accessory cells transduced with the CD gene; one type of non-cancerous accessory cell found in primary bone cancer and breast cancer metastases to bone is the osteoclast. This manuscript determines if osteoclast precursor cells, transduced with the CD gene, can function as a gene delivery system capable of killing cancer cells. An osteoclast precursor cell line (RAW 264.7, RAW) and authentic bone marrow-derived osteoclast precursor cells were transduced with a retroviral vector containing the cytosine deaminase fusion gene (NCD) composed of the human nerve growth factor receptor and CD genes. RAW cells and bone marrow-derived osteoclast precursor cells transduced with NCD expressed NCD protein and converted 5FC to 5FU. Treatment of NCD-transduced osteoclast precursor cells with the 5FC prodrug resulted in significant killing in vitro. NCD-transduced osteoclasts were co-cultured with either DsRed2-labeled sarcoma cells (2472-DSR) or green fluorescent protein (GFP)-labeled breast cancer cells (GFP-4T1). Treatment of the NCD osteoclast/tumor cell co-cultures with 5FC resulted in bystander killing of 2472-DSR cells (P < 0.006) and GFP-4T1 cells (P < 0.004). These findings demonstrate that NCD-transduced osteoclasts can promote killing of cancer cells and introduce the exciting possibility for developing osteoclast-mediated, CD-based treatment of primary bone cancers and breast cancer metastases to bone.
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PMID:Osteoclasts direct bystander killing of cancer cells in vitro. 1613 79

Donor T lymphocytes genetically engineered to express a "suicide gene" to facilitate negative selection represent a promising strategy for the management of graft-versus-host disease occurring after allogeneic hematopoietic cell transplantation (HCT). For this purpose, the herpes simplex virus thymidine kinase (HSV-tk) gene, although well studied, has limitations. Cytosine deaminase (CD), an alternative gene for negative selection, converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). Sensitivity of cells to 5-FU can be further increased by expression of uracil phosphoribosyltransferase (UPRT), which catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate. By using a chimeric gene (NG/CD) expressing the truncated human nerve growth factor receptor (NGFR) for positive selection fused to the Saccharomyces cerevisiae CD gene, we investigated strategies to achieve optimal T cell eradication by CD and UPRT expression, utilizing a single retroviral vector. Three vector strategies were compared on the basis of NGFR expression by flow cytometry, western analysis, and enzymatic activity. A construct (NG/CDiU) expressing UPRT and NG/CD, using a bicistronic message, provided the greatest UPRT activity and killing, reducing the lethal dose of 5-FC sufficient to eradicate 90% of cells from 38.7 microg/ml (300 microM) (NG/CD expression alone) to 0.13 microg/ml (1 microM). This approach provides an effective alternative to the HSV-tk system for eradication of donor T lymphocytes after allogeneic HCT.
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PMID:Coexpression of the uracil phosphoribosyltransferase gene with a chimeric human nerve growth factor receptor/cytosine deaminase fusion gene, using a single retroviral vector, augments cytotoxicity of transduced human T cells exposed to 5-fluorocytosine. 1671 9