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

Previously, we reported that adenoviral vectors carrying the carcinoembryonic antigen (CEA) promoter sequences to direct the Echerichia coli beta-galactosidase gene (AdCEA-lacZ) or cytosine deaminase (CD) gene (AdCEA-CD) confer selective gene expression on a CEA-positive gastric cancer cell line (MKN45) in vitro. Here, adenovirus-mediated tumor-specific gene therapy for CEA-positive gastric carcinoma in vivo was investigated. Using an animal model with i.p. disseminated MKN45 tumors, adenovirus-mediated tumor-specific transgene expression and therapeutic efficacy were analyzed. After an i.p. injection of AdCEA-lacZ, beta-galactosidase activity was confined to tumor xenografts. Moreover, CD mRNA was expressed exclusively in MKN45 tumor xenografts after infection with AdCEA-CD, despite the fact that an adenovirus-mediated transfer of CD DNA was detected in all tissues tested. In contrast, CD mRNA was detected not only in tumor xenografts but also in other organs of mice infected with AdCA-CD, in which CD gene expression is governed by an ubiquitous promoter. Suppression of tumor growth and prolongation of survival were noted in tumor-bearing mice treated with AdCEA-CD and 5-fluorocytosine (5FC) without observable adverse effects. In contrast, significant hepatic toxicity was noted in animals treated with AdCA-CD. These results reveal that the CEA promoter restricts CD gene expression to CEA-positive tumor cells in the adenoviral context in vivo, along with the beneficial therapeutic effects of 5FC treatment, suggesting the i.p. AdCEA-CD/5FC system may provide a novel approach to treatment of i.p. disseminated gastric cancer.
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PMID:In vivo selective gene expression and therapy mediated by adenoviral vectors for human carcinoembryonic antigen-producing gastric carcinoma. 933 Oct 89

We have recently isolated carcinoembryonic antigen (CEA) promoter regions consisting of 419 bp and 204 bp from CEA-producing human colorectal carcinoma (CRC). We constructed CEA419/CD and CEA204/CD retroviruses carrying the bacterial cytosine deaminase (CD) gene directed by the CEA promoter regions. pCD2 retroviruses carrying the CD gene directed by the retrovirus long terminal repeat promoter were also used. CEA419/CD or CEA204/CD retrovirus-infected CRC cells were found to be susceptible to 5-fluorocytosine (5-FC), while non-CRC cells infected with the same retroviruses were not. CD-transduced CRC xenografts in nude mice were sensitive to 5-FC treatment, resulting in arrest of tumor growth. When mice with intraperitoneally disseminated CRCs were given intraperitoneal injections of CEA419/CD retrovirus-producing cells followed by 5-FC treatment, significantly prolonged survival rates were observed compared with animals injected with pCD2 retrovirus-producing cells followed by 5-FC treatment. Importantly, bone marrow suppression was not observed in animals injected with CEA419/CD retrovirus-producing cells and 5-FC, while profound bone marrow suppression was observed in those injected with pCD2 retrovirus-producing cells and 5-FC. These results indicate that effective and safe in vivo gene therapy for advanced CRC may be feasible by transferring the CD gene controlled by the CEA promoter followed by 5-FC treatment.
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PMID:Effective and safe gene therapy for colorectal carcinoma using the cytosine deaminase gene directed by the carcinoembryonic antigen promoter. 1034 79

We constructed the CEA419/CD retrovirus vector carrying the cytosine deaminase (CD) gene directed by the carcinoembryonic antigen (CEA) promoter. pCD2 retrovirus vector carrying the CD gene directed by the retrovirus long terminal repeat promoter was also used. When mice bearing intraperitoneally disseminated colorectal carcinomas (CRCs) were infused intraperitoneally with pCD2 or CEA419/CD retrovirus-producing cells, a CD fragment was detected in CRCs and bone marrow cells. It was shown that the CD gene was expressed both in CRCs and in the bone marrow of animals infused with pCD2 retrovirus-producing cells, while the CD gene was expressed solely in CRCs of animals infused with CEA419/CD retrovirus-producing cells. These results indicate that the use of a tumor-selective promoter may warrant the safety of in vivo gene therapy using suicide genes.
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PMID:In vivo gene transfer of a suicide gene under the transcriptional control of the carcinoembryonic antigen promoter results in bone marrow transduction but can avoid bone marrow suppression. 1037 1

We isolated a 204-base pair carcinoembryonic antigen (CEA) promoter core region from a CEA-producing human colorectal carcinoma (CRC) and constructed retrovirus vectors carrying the expression cassette consisting of the CEA promoter core region and the cytosine deaminase (CD) gene. pCD2 retrovirus carrying the CD gene directed by the retrovirus long terminal repeat promoter served as a control vector. An in vitro study showed that the CEA promoter conferred CEA-producing cell-selective CD expression, specifically when the CD expression cassette was inserted into the 3' long terminal repeat of the retrovirus vector. CD-modified CRC xenografts in nude mice were sensitive to 5-fluorocytosine and caused a profound bystander effect on the unmodified CRC. When nude mice harboring intraperitoneally disseminated CRCs were injected intraperitoneally with the CD expression cassette-carrying retrovirus-producing cells, CD transduction into the disseminated CRCs and bone marrow (BM) was observed. CD expression was, however, restricted to CRCs, and it was observed in both CRCs and BM of mice injected with pCD2 retrovirus-producing cells, resulting in better therapeutic outcomes without BM suppression. These results indicate that effective and safe in vivo gene therapy for CRC may be feasible by transferring the CD gene controlled by the CEA promoter core region.
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PMID:Analysis of the human carcinoembryonic antigen promoter core region in colorectal carcinoma-selective cytosine deaminase gene therapy. 1060 54

Utilization of molecular biology techniques offers attractive options in nuclear medicine for improving cancer imaging and therapy with radiolabeled peptides. Two of these options include utilization of phage-panning to identify novel tumor-specific peptides or single chain antibodies and gene transfer techniques to increase the number of antigen/receptor sites expressed on malignant cells. Our group has focused on the latter approach for improving radiolabeled peptide imaging and therapy. The most widely used gene transfer vectors in clinical gene therapy trials include retrovirus, cationic lipids, and adenovirus. We have utilized adenovirus vectors for gene transfer because of their ability to accomplish efficient in vivo gene transfer. Adenovirus vectors encoding the genes for a variety of antigens/receptors (carcinoembryonic antigen, gastrin-releasing peptide receptor, somatostatin receptor subtype 2 (SSTr2)) have all shown that their expression is increased on cancer cells both in vitro and in vivo following adenovirus infection. Of particular interest has been the adenovirus encoding for SSTr2 (AdCMVSSTr2). Various radioisotopes have been attached to somatostatin analogues for imaging and therapy of SSTr2-positive tumors both clinically and in animal models. The use of these analogues in combination with AdCMVSSTr2 is a promising approach for improving the detection sensitivity and therapeutic efficacy of these radiolabeled peptides against solid tumors. In addition, we have proposed the use of SSTr2 as a marker for imaging the expression of another cancer therapeutic transgene (e.g. cytosine deaminase, thymidine kinase) encoded within the same vector. This would allow for non-invasive monitoring of gene delivery to tumor sites.
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PMID:Gene transfer strategies for improving radiolabeled peptide imaging and therapy. 1110 86

Yeast cytosine deaminase (yCD)-based gene therapy offers the potential for selective production of the cytotoxic and radiosensitizing drug 5-fluorouracil (5-FU) from the benign prodrug 5-fluorocytosine within colorectal cancers. Although previous attempts to target therapy to colorectal cancer using the carcinoembryonic antigen (CEA) promoter have demonstrated specificity, this has been achieved at the cost of 10- to 300-fold loss in activity compared with strong but nonspecific rous sarcoma virus (RSV) or cytomegalovirus promoters. We developed a highly specific and active gene transfer method for colorectal cancer using CEA under control of a promoter-enhancer. We compared the RSV promoter-derived with the CEA promoter-enhancer-derived transgene expression in 10 different cell lines with differing CEA status. We found that the transgene expression resulting from both transient transfection and adenoviral infection with the CEA promoter-enhancer was as strong as the RSV promoter while maintaining specificity for CEA-producing cell lines. For instance, when we compared yCD expression between LoVo (CEA+) and human fibroblast (CEA-), we found a 30-fold-increased yCD expression in LoVo cells from CEA-enhancer adenovirus although there was no difference in the yCD expression between the cell lines when infected with RSV/yCD virus. This specificity was also achieved while maintaining a higher yCD enzyme activity than we obtained with RSV/yCD adenovirus in an HT-29 intrahepatic tumor model. We then compared the response of HT-29 xenografts to treatment with 5-fluorocytosine and yCD adenovirus driven by either the RSV or the CEA promoter-enhancer and found similar tumor growth inhibition. These findings suggest that the CEA promoter-enhancer strategy confers specificity while preserving activity and is worth exploring in additional animal and, potentially, clinical trials.
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PMID:High and selective expression of yeast cytosine deaminase under a carcinoembryonic antigen promoter-enhancer. 1195 93

The possibility of tumor-specific suicidal gene therapy of colorectalcarcinoma was investigated using carcinoembryonic antigen (CEA) -positive human colorectal carcinoma cell line LoVo and CEA-negative HeLa cell line as a model. After confirming cellular specificity of cea promoter by CAT assay, eukaryotic expression plasmid pCEACD was constructed in which the expresstion of E. coli cytosine deaminase (CD) gene was under the control of cea promoter. The expression of CD gene increased the sensitivity of LoVo cells to 5-fluorocytosine (5FC) by 750 fold, while the sensitivity of HeLa cells to 5FC was increased by only 7.5 fold. These results suggest that the expression of CD gene driven by cea promoter specifically killed CEA-positive colorectal carcinoma cells. Transmission electron microscopy and DNA fragmentation assay demonstrate that CD/5FC system induced apoptosis in LoVo cells.
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PMID:Use of Carcinoembryonic Antigen Gene Promoter in Colorectal Carcinoma-specific Suicidal Gene Therapy. 1217 89

A major potential limitation to the success of enzyme prodrug gene therapy is the toxicity that could result from gene expression in normal tissues. In this study, we investigated the use of an enhanced human carcinoembryonic antigen (CEA) promoter for yeast cytosine deaminase (yCD), which converts 5-fluorocytosine to 5-fluorouracil, to increase targeting while maintaining activity both in cell culture and in nude rats bearing intrahepatic xenografts. We found that an enhanced CEA-yCD adenoviral vector can achieve significantly greater yCD expression in CEA-expressing colon carcinoma cell lines (LoVo, HT29, and CaCo2) compared with a nonspecific Rous sarcoma virus-yCD virus. In contrast, infection with CEA-yCD led to lower or equivalent yCD expression in normal hepatocytes or fibroblasts compared with that produced by the RSV-yCD. Adenovirus administered in the portal vein or the hepatic artery of nude rats bearing intrahepatic LoVo colon carcinomas could mediate beta-galactosidase expression equally in liver and tumors under the control of cytomegalovirus, a nonspecific promoter. However, infusion of CEA-yCD virus markedly increased yCD expression in tumors over normal liver (>4-fold) measured both by levels of mRNA and yCD activity. Moreover, the efficiency of 5-fluorocytosine conversion into 5-fluorouracil in tumors was significantly higher than that in normal liver ( approximately 3-fold) in rats receiving portal venous viral infusion of CEA-yCD and subsequent 5FC treatment. Thus, an enhanced CEA promoter can preferentially stimulate yCD gene expression in CEA-expressing cells in vivo. Such tumor-specific expression should prove useful in colorectal cancer gene therapy to achieve selective prodrug conversion in tumors.
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PMID:Regional delivery and selective expression of a high-activity yeast cytosine deaminase in an intrahepatic colon cancer model. 1256 11

Peritoneal dissemination is a common end-stage complication of pancreatic cancer for which novel therapeutic modalities are actively investigated, as there is no current effective therapy. Thus, we evaluated, in a mouse model of pancreatic peritoneal carcinomatosis, the therapeutic potential of a novel nonviral gene therapy approach consisting of bis-guanidinium-tren-cholesterol (BGTC)-mediated lipofection of a combined suicide gene system. Human BxPC-3 pancreatic cells secreting the carcinoembryonic antigen (CEA) tumor marker were injected into the peritoneal cavity of nude mice. After 8 days, intraperitoneal (i.p.) lipofection was performed using BGTC/DOPE cationic liposomes complexed with plasmids encoding the two prodrug-activating enzymes Herpes Simplex Virus thymidine kinase and Escherichia coli cytosine deaminase, the latter being expressed from a bicistronic cassette also encoding E. coli uracil phosphoribosyltransferase. Administration of the lipoplexes was followed by treatment with the corresponding prodrugs ganciclovir and 5-fluorocytosine. The results presented herein demonstrate that BGTC/DOPE liposomes can efficiently mediate gene transfection into peritoneal tumor nodules. Indeed, HSV-TK mRNA was detected in tumor nodule tissues by semiquantitative reverse transcription-polymerase chain reaction analysis. In addition, green fluorescent protein (GFP) fluorescence and X-gal staining were observed in the peritoneal tumor foci following lipofection of the corresponding EGFP and LacZ reporter genes. These expression analyses also showed that transgene expression lasted for about 2 weeks and was preferential for the tumor nodules, this tumor preference being in good agreement with the absence of obvious treatment-related toxicity. Most importantly, mice receiving the full treatment scheme (BGTC liposomes, suicide genes and prodrugs) had significantly lower serum CEA levels than those of the various control groups, a finding indicating that peritoneal carcinomatosis progression was strongly reduced in these mice. In conclusion, our results demonstrate the therapeutic efficiency of BGTC-mediated i.p. lipofection of a combined suicide gene system in a mouse peritoneal carcinomatosis model and suggest that BGTC-based prodrug-activating gene therapy approaches may constitute a potential treatment modality for patients with peritoneal carcinomatosis and minimal residual disease.
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PMID:Combined suicide gene therapy for pancreatic peritoneal carcinomatosis using BGTC liposomes. 1468 23


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