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 gene encoding cytosine deaminase (CD) has been expressed in the human colorectal carcinoma cell line WiDr. Metabolism studies confirm that tumor cells expressing CD convert the very nontoxic prodrug 5-fluorocytosine (5FCyt) to 5-fluorouracil (5FUra) and 5FUra metabolites. Tumor xenografts composed of CD-expressing cells can selectively generate tumor levels of > 400 microM 5FUra when the host mouse is dosed with nontoxic levels of 5FCyt. The selective metabolic conversion of 5FCyt to 5FUra in CD-expressing tumor cells results in the inhibition of thymidylate synthase and incorporation of 5FUra into RNA. 5FUra is also liberated into the surrounding environment when CD-expressing tumor cells are treated with 5FCyt. The liberated 5FUra is able to kill neighboring, non-CD-expressing tumor cells in vitro and in vivo. Most importantly, when only 2% of the tumor mass contains CD-expressing cells (98% non-CD-expressing cells), significant regressions in all tumors are observed when the host mouse is dosed with nontoxic levels of 5FCyt.
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PMID:Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. 805 98

A human colorectal carcinoma cell line, WiDr, was genetically engineered to express the nonmammalian enzyme, cytosine deaminase (CD). Expression of CD in WiDr cells (WiDr/CD) did not alter the growth rate of these cells when grown in vitro or as solid tumor xenografts in nude mice. However, expression of CD did increase the sensitivity of these cells to the nontoxic prodrug, 5-fluorocytosine (FCyt), decreasing the 50% inhibitory concentration for FCyt from 26,000 microM in parental WiDr cells to 27 microM in WiDr/CD cells. The increase in sensitivity to FCyt in WiDr/CD cells was the result of the CD-mediated conversion of FCyt to 5-fluorouracil (FUra) and subsequent FUra anabolites. The half-life of the prodrug, FCyt, was determined to be approximately 40 min in nude mice. A single i.p. injection of 500 mg FCyt/kg body weight resulted in a transient FCyt plasma level of approximately 4000 microM while osmotic minipumps or constant tail vein infusions of FCyt achieved continual FCyt plasma levels of 5 microM and 50 microM, respectively, with no overt signs of toxicity. Significant antitumor effects were observed in nude mice bearing tumors derived from WiDr/CD cells when these animals were given 500 mg FCyt/kg i.p. for 10 consecutive days. These antitumor effects were demonstrated by decreases in tumor growth rate, tumor size, tumor weight, and thymidine incorporation into tumor DNA. This antitumor effect was significant but less profound if FCyt was administered by constant tail vein infusion. WiDr and WiDr/CD cells were very sensitive to FUra in vitro (50% inhibitory concentration approximately 5 microM). However, no significant antitumor effects were observed in nude mice bearing tumors derived from either WiDr or WiDr/CD cells when these animals were treated with various doses of FUra. Taken collectively, these data indicate that nontoxic plasma levels of FCyt can be attained which can produce profound antitumor effects on tumors engineered to express CD and that these antitumor effects are significantly better than those that can be achieved using FUra. These positive data support the continued development of a gene therapy approach to colorectal carcinoma involving the selective expression of CD in colorectal tumors with subsequent administration of FCyt.
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PMID:In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. 840 37

We have developed a new approach involving gene therapy for the treatment of primary and metastatic tumors in the liver. As a first step toward the development of this gene therapy treatment for metastatic colorectal carcinoma, the Escherichia coli gene that encodes cytosine deaminase (CD) (EC 3.5.4.1) has been cloned. By using positive genetic selection, a plasmid carrying a 10.8-kilobase BamHI/EcoRI DNA insert was isolated that had CD enzymatic activity. Genetic screening, followed by enzymatic assays, identified a 3-kilobase DNA fragment that retained CD activity. Deamination of cytosine and 5-fluorocytosine (5-FC) by cloned CD was demonstrated. DNA and protein sequencing identified an open reading frame of 427 amino acids that encodes CD. To demonstrate that expression of CD in eukaryotic cells allows metabolism of the nontoxic prodrug 5-FC to the toxic metabolite 5-fluorouracil, CD was cloned into a eukaryotic expression vector and transfected into a human colorectal carcinoma cell line. Growth inhibition studies showed a shift in the IC50 for 5-FC from 17,000 microM in the parental cell line to 30 microM in cells expressing CD.
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PMID:A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase. 845 Aug 32

To evaluate the hypothesis that regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene (AdCMV.CD) together with systemic 5-FC could suppress the growth of metastatic colon cancer in the liver, the AdCMV.CD vector was injected 0.8-1 cm from the site of a human colon cancer tumor in the livers of nude mice. The growth of the human colon cancer cells was quantified by dot blot analysis of genomic DNA extracted from tumor-bearing liver, hybridized with a human-specific Alu probe. The combination of regional AdCMV.CD plus systemic 5-fluorocytosine (5-FC) suppressed the growth of the metastatic tumors over the 21 days of evaluation following vector administration. Histologic evaluation showed necrosis at the site of the tumor in the livers of mice treated with AdCMV.CD/5-FC, but not in control groups. Evaluation of the potential toxicity of AdCMV.CD plus 5-FC on the normal liver showed only mild, self-limited dose-related inflammation, with no deaths. These data suggest that the regional administration of AdCMV.CD together with systemic 5-FC may be a safe and effective strategy to suppress the growth of metastases of colorectal carcinoma in the liver.
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PMID:Regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene to provide local activation of 5-fluorocytosine to suppress the growth of colon carcinoma metastatic to liver. 886 57

Certain species of anaerobic bacteria have been shown to localise and germinate specifically in the hypoxic regions of tumours, resulting in tumour lysis. We propose an innovative approach to cancer gene therapy in which genetically engineered anaerobic bacteria of the genus Clostridium are used to achieve tumour-specific gene delivery. Our strategy involves enzyme/prodrug therapy, in which the Escherichia coli enzyme cytosine deaminase is used to convert the non-toxic prodrug 5-fluorocytosine to the active chemotherapeutic agent 5-fluorouracil. The E. coli gene encoding cytosine deaminase has been cloned into a clostridial expression vector and transformed into Clostridium beijerinckii, resulting in constitutive expression of cytosine deaminase and significant levels of active enzyme in the bacterial medium. When added to an in vitro clonogenic survival assay, supernatant from clostridia expressing cytosine deaminase increased the sensitivity of murine EMT6 carcinoma cells to 5-fluorocytosine approximately 500-fold. This high level of prodrug activation, combined with the specificity of clostridia for hypoxic regions of tumours, indicates a potential use in cancer gene therapy.
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PMID:Anaerobic bacteria as a delivery system for cancer gene therapy: in vitro activation of 5-fluorocytosine by genetically engineered clostridia. 886 65

Gene therapy combined with radiation therapy to enhance selectively radiation cytotoxicity in malignant cells represents a new approach for cancer treatment. We investigated the efficacy of adenoviral (Ad5)-directed cytosine deaminase/5-fluorocytosine (CD/5-FC) enzyme/prodrug gene therapy to enhance selectively the tumoricidal action of ionizing radiation in human cancer xenografts derived from a human squamous carcinoma cell line (SQ-20B). Tumor xenografts grown in hindlimbs of nude mice were transfected with an adenoviral vector (Ad.CMV.CD) containing the cytosine deaminase (CD) gene under the control of a cytomegalovirus (CMV) promoter. Mice were injected i.p. with 800 mg/kg of 5-FC for 12 days, and tumors were treated with fractionated radiation at a dose of 5 Gy/day to a total dose of 50 Gy. In larger tumors with a mean volume of 1069 mm3, marked tumor regression to 11% of the original tumor volume was observed at day 21 (P = 0.01). The volumetric regression of smaller tumors with a mean volume of 199 mm3, which received the same combined treatment protocol, was significant at day 12 (P = 0.014). However, unlike large tumors, regression of the smaller tumors continued until day 36 (P = 0.01), with 43% cured at day 26. No cures or significant volumetric reduction in size was observed in tumors treated with radiation alone; Ad.CMV.CD with or without radiation; or with Ad.CMV.CD and 5-FC. These results suggest that the CD/5-FC gene therapy approach is an effective radiosensitizing strategy and may lead to substantial improvement in local tumor control that would translate into improved cure rates and better survival.
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PMID:Virally directed cytosine deaminase/5-fluorocytosine gene therapy enhances radiation response in human cancer xenografts. 933 Oct 76

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

To develop a suitable suicide gene/prodrug therapy for the treatment of thyroid carcinomas, the relative therapeutic efficacy of four different suicide gene/prodrug combinations was compared in thyroid carcinomas in vitro. Herpes simplex virus thymidine kinase and ganciclovir (HSV-TK/GCV), Escherichia coli cytosine deaminase and 5-fluorocytosine (CD/5FC), E coli nitroreductase and CB1954 (NTR/CB1954), and human deoxycytidine kinase and cytosine arabinoside (dCK/AraC) were employed. The suicide genes were transduced into two thyroid carcinoma cell lines with retroviral vectors in which all the suicide genes were under the control of the same promoter. When the relative efficacy of four suicide gene/prodrugs was compared with therapeutic index and degree of bystander effect, we found a clear dissociation between these two parameters. Thus, HSV-TKIGCV demonstrated the widest therapeutic index, while CD/5FC and NTR/CB1954 showed the stronger bystander effect than HSV-TK/GCV. dCK/AraC had little efficacy. Advantages and limitations of each suicide gene/prodrug combinations are discussed.
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PMID:Treatment of thyroid carcinoma cells with four different suicide gene/prodrug combinations in vitro. 967 64

Recently, use of the suicide gene, cytosine deaminase (CD), has shown a selective antitumor activity of 5-fluorocytosine (5-FC) on human colorectal carcinoma cells grown in vitro and in vivo. We hypothesized that the radiosensitivity of human colorectal carcinoma cells transduced with a retroviral vector encoding the bacterial CD gene would be selectively enhanced by the nontoxic prodrug 5-FC. The radiobiological rationale of using suicide gene therapy is based on the fact that a toxic metabolite of 5-FC, 5-fluorouracil, is a well-known radiation enhancer for the treatment of gastrointestinal and other tumors. 5-FC was found to enhance selectively the radiation cytotoxicity of human colorectal carcinoma cells expressing the CD gene. Colorectal carcinoma cells transduced with the CD gene (WiDr-CD) were highly sensitive to radiation compared with parental cells (WiDr) when exposed to 20 microgram/ml 5-FC for 72 h prior to irradiation. The sensitization enhancement ratio was 2.38. This magnitude of radiation enhancement is comparable to that obtained with 5-fluorouracil. These results suggest that the addition of radiation would substantially improve the therapeutic potential of CD gene therapy for the treatment of locally advanced colorectal carcinomas.
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PMID:Radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells in culture transduced with cytosine deaminase gene. 981 90

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


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