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)

The 5' sequences from the human carcinoembryonic antigen gene (CEA) were analyzed using luciferase reporter gene assays. This analysis identified important cis-acting sequences needed for selective expression in CEA-positive cells. Over 50 CEA/luciferase reporter clones were constructed and analyzed in two CEA-positive and two CEA-negative cell lines. The CEA sequences analyzed extended from the translational start to 14.5 kb 5' of the CEA gene. A 408-bp region from the CEA 3' untranslated region was also examined for its effect on reporter gene activity. The CEA promoter was located between bases -90 and +69 of the transcriptional start site. Sequences between -41 and -18 were essential for expression from the CEA promoter. Multimerization of sequences between -89 and -40 resulted in copy number-related increases in both expression level and selectivity for CEA-positive cells. Two upstream regions of CEA, -13.6 to -10.7 kb or -6.1 to -4.0 kb, when linked to the multimerized promoter led to high-level, selective expression in CEA-positive cell lines. Several CEA/luciferase constructs demonstrated 80- to 120-fold higher expression in CEA-positive cell lines compared to expression in CEA-negative Hep3B cells. The expression from these constructs was quite strong in CEA-positive cells, being two- to four-fold higher than an SV40 enhancer/promoter construct. The most promising CEA transcriptional regulatory sequences were used to regulate the expression of cytosine deaminase (CD) in stable cell lines. The expression of CD was assessed directly by an enzymatic assay and indirectly by determining the in vitro IC50 to 5-fluorocytosine (5FC). The chimeric gene pCEA/CD-145 displayed the desired expression spectrum--high-level expression in the CEA-positive cells and low-level expression in CEA-negative cells. CD expression from this chimera correlated well with the expression of the endogenous CEA gene. Treatment of mice bearing NCI H508 pCEA/CD-145 tumor xenografts with 5FC lead to significant antitumor effects in vivo. The CEA/CD chimeric gene should be useful for tumor-specific suicide gene therapy of CEA-positive tumors.
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PMID:Transcriptional regulatory sequences of carcinoembryonic antigen: identification and use with cytosine deaminase for tumor-specific gene therapy. 757 7

Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and effective in vitro transfection method may also be applicable to in vivo delivery of target genes to the vascular wall to inhibit SMC proliferation.
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PMID:Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells. 880 Apr 93

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

Prostate-specific membrane antigen (PSMA) is a type-2 membrane protein expressed in the prostate, and it is highly expressed in metastatic or poorly differentiated adenocarcinomas. Moreover, PSMA expression is upregulated by androgen deprivation. These advantages make PSMA a useful target for prostate cancer therapy, especially in combination with conventional hormonal treatment. We recently reported that a prostate-specific enhancer is present in the third intron of the PSMA gene. In this study, we have further analyzed the activity of PSMA promoter/enhancer in prostate cancer cells and cells of other tissue origins (breast cancer MCF-7, lung cancer H157, and colorectal cancer HCT8 cells), and we have examined whether this construct could be used for efficient expression of the suicide gene, cytosine deaminase (CD), in vivo. The PSMA promoter/enhancer expressed the luciferase reporter gene in the prostate cancer lines LNCaP and C4-2, with 8- to 20-fold higher expression than the simian virus 40 promoter/enhancer, although it was inactive in the other cell lines. This construct efficiently drove the suicide gene CD, sensitizing C4-2 cells to 5-fluorocytosine (5-FC) with the inhibitory concentration (IC(50)) <300 micromol/L in vitro. Athymic male nude mice bearing the transfected C4-2 cells were treated with intraperitoneal injections of either 5-FC (600 mg/kg) twice a day or saline solution for 3 weeks. C4-2 cell tumors were eliminated by 5-FC when they were expressing our therapeutic construct carrying CD under the regulatory control of the PSMA promoter/enhancer. Our results show the in vivo utility of the PSMA promoter/enhancer in a gene therapy situation targeting prostate cancer.
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PMID:In vivo suicide gene therapy model using a newly discovered prostate-specific membrane antigen promoter/enhancer: a potential alternative approach to androgen deprivation therapy. 1150 68

Colorectal cancer can metastasize to the liver, but remain liver confined for years. A critical step in developing treatments for intrahepatic cancer involves assessment in an orthotopic intrahepatic model. The purpose of this study was to develop a noninvasive intrahepatic tumor model to study the efficacy of 5-flucytosine/yeast cytosine deaminase (5FC/yCD)-based gene therapy for liver tumors. Luciferase expressing human colorectal carcinoma (HT-29luc) cells were generated by retroviral infection and implanted in the left liver lobe of nude mice. The bioluminescence was measured every week for a period of 1 month, then animals were killed and tumors were measured by calipers. After we found a correlation between photon counts and tumor size, animals were implanted with tumors composed of either 0%, 10%, or 100% yCD/HT-29luc cells, and treated with 5FC. Tumor bioluminescence was measured during treatment and tumor histology examined at the time of death. We found that 5FC caused significant regression of yCD expressing tumors. Furthermore, visible tumors at the time of death, which emitted little bioluminescence, contained little or no viable tumor. We then developed an adenoviral vector for yCD. Intraperitoneal administration of adenovirus containing yCD led to the production of yCD enzyme within intrahepatic tumors. These results suggest that (1) intrahepatic cancer responds to 5FC when cells express yCD; (2) the luciferin-luciferase system permits non-invasive real time imaging of viable intrahepatic cancer; and (3) this system can be used to carry out gene therapy experiments using yCD adenovirus.
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PMID:The potential of 5-fluorocytosine/cytosine deaminase enzyme prodrug gene therapy in an intrahepatic colon cancer model. 1208 Mar 78

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase, is transcriptionally upregulated in more than 90% of tumor cells. It may be used as a tool for driving a gene to kill tumors specifically. To test this idea, luciferase reporter gene was used and the results showed that hTERT promoter could restrict the gene expression in the telomerase-positive tumor cells. A tumor-specific expression plasmid phTERT-CD was constructed, in which the E. coli cytosine deaminase (CD) gene was controlled by the hTERT promoter. A colorectal cancer cell line (LoVo) and a normal amnion cell line (WISH) were transfected by this plasmid. It was shown that the expression of the CD gene increased the sensitivity of LoVo cells to the prodrug, 5-fluorocytosine (5FC), over 800-fold, while the sensitivity of WISH cells to 5FC was increased only 6-fold. Mixed cell experiments showed a strong "bystander effect" on CD-negative cells. Furthermore, a significant anti-tumor effect of the phTERT-CD/5FC system was observed in nude mice bearing mammalian carcinoma induced by s.c. inoculation of LoVo cells when the mice were given 250 mg/kg 5FC twice a day for 10 consecutive days. These results indicated that hTERT promoter could target the suicidal effect of CD gene to tumor cells, and therefore, may be a novel and promising targeting approach to the treatment of cancer.
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PMID:Cancer-specific killing by the CD suicide gene using the human telomerase reverse transcriptase promoter. 1216 15

Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD) along with an optical reporter gene (luciferase). Following intratumoral injection of the vector into orthotopic 9 L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC) gene therapy. Significant increases in mean tumor diffusion values were also observed during treatment with 5FC. Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass. In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging for assessment of gene expression and therapeutic efficacy.
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PMID:Molecular imaging of gene expression and efficacy following adenoviral-mediated brain tumor gene therapy. 1292 Aug 60

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.
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PMID:Use of the EGP-2/Ep-CAM promoter for targeted expression of heterologous genes in carcinoma derived cell lines. 1524 30

Endostatin, an angiogenesis inhibitor tested in multiple clinical trials, selectively targets neovascular endothelial cells, suppressing tumor growth. To enhance the therapeutic efficacy of endostatin, we fused endostatin with cytosine deaminase, which converts a prodrug 5-flucytosine into a cytotoxic 5-fluorouracil. This therapeutic strategy was developed based on the observation that the endostatin-green fluorescence protein gene and endostatin-luciferase gene selectively target to endothelial cells in vitro and to the tumor site in vivo, respectively. When we used the endostatin-cytosine deaminase fusion protein to treat s.c. grafted tumors or experimental metastasis tumors, our results showed that endostatin-cytosine deaminase treatment provided stronger tumor growth suppression and increased mean survival time of the mice compared with the treatments of endostatin alone, cytosine deaminase alone, or endostatin plus cytosine deaminase. The endostatin-cytosine deaminase protein significantly inhibited the growth of endothelial cells and preferentially induced tumor cell apoptosis. This endostatin-cytosine deaminase fusion approach opens an avenue for cancer-targeting therapy.
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PMID:Endostatin-cytosine deaminase fusion protein suppresses tumor growth by targeting neovascular endothelial cells. 1639 52

Hypoxia, a hallmark of many solid tumors, is associated with angiogenesis and tumor progression. It activates a signal cascade that culminates in the stabilization of hypoxia-inducible factor-1 (HIF-1) transcription factor and activation of genes that possess hypoxia response elements. The loss of tumor suppressors such as p53 has been shown to stabilize HIF-1alpha, which is overexpressed in the majority of human cancers, and its over-expression correlates with poor prognosis and treatment failure. Here we constructed hypoxia-inducible promoters and examined their activities in murine and human cancer cells with variable p53 status. Loss of p53 function in cancer cells resulted in increased HIF-1-dependent transcriptional activity. To investigate the feasibility of exploiting the hypoxic tumor microenvironment for targeted gene therapy of cancer, we constructed retroviral vectors harboring luciferase or Escherichia coli cytosine deaminase (CD) genes under the control of the hypoxia-inducible promoter. Murine Lewis lung carcinoma (LL2) cells carrying defective p53, when retrovirally transduced with the hypoxia-inducible promoter-driven luciferase gene under hypoxic conditions, increased luciferase reporter gene expression in vitro and in vivo. Significant antitumor effects were achieved in mice bearing LL2 tumors that expressed CD driven by a hypoxia-inducible promoter after treatment with 5-fluorocytosine. These results suggest the potential applications of suicide genes, such as the CD gene, under the control of hypoxia-inducible promoters for cancer gene therapy, which may target efficiently to hypoxic regions of tumors with p53 mutations.
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PMID:Hypoxia-induced cytosine deaminase gene expression for cancer therapy. 1718 54


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