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:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.
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PMID:Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference. 1524 53

B-cell lymphomas arising in lymph nodes and spleen of aging mice deficient in the Ung DNA glycosylase were recovered, dispersed, grown in short-term culture, and CD19-positive B-cells retrieved and analysed. Several tumors as well as controls only expressed detectable amounts of the Aid deaminase after mitogenic stimulation, as estimated by real-time PCR of transcripts. However, one unusually large lymph node tumor expressed a high level of Aid constitutively. This particular tumor also showed a substantially increased mutation frequency in the Aid gene itself as well as in the bcl-6 and c-myc genes, but not in the p53 gene, consistent with aberrant somatic hypermutation. Other B-cell lymphomas from Ung(-/-) mice exhibited a modest increase in mutation frequency.
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PMID:Mutation frequencies and AID activation state in B-cell lymphomas from Ung-deficient mice. 1573 13

Housekeeping genes are commonly used as endogenous references in quantitative RT-PCR. Ideally these genes are constitutionally expressed by all cell types and do not vary under experimental conditions. Tissues of 9 normal testes and 22 classical pure seminoma were obtained for RNA-extraction. Real-time RT-PCR was used to examine the mRNA-expression of ubiquitin C, beta-actin, GAPDH, 18S ribosomal RNA (18S rRNA) and porphobilinogen-deaminase (PBGD). Additionally, 3 normal testicular tissues and 39 seminoma, including 1 normal testis and 17 seminoma of the RT-PCR group, were utilized for microarray analyses. Ubiquitin C (protein degradation) was down-regulated, GAPDH (carbohydrate metabolism), beta-actin (cytoskeleton), 18S rRNA (ribosome) and PBGD (porphyrin metabolism) were up-regulated in seminoma. A normalization of the target gene data with up-regulated housekeeping genes would equalize or underestimate up-regulated data and overestimate down-regulated data. We demonstrate that none of the investigated housekeeping genes is suitable for normalization of the target gene RT-PCR data, but may be essential for tumor metabolism in human seminoma. Further, we developed a standardization strategy, which is applicable to many experimental investigations.
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PMID:Standardization strategy for quantitative PCR in human seminoma and normal testis. 1582 5

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

Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.
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PMID:In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant. 1623 Apr 16

Histone deacetylases (HDACs) are among the most promising targets in cancer therapy. However, structural information greatly enhancing the design of HDAC inhibitors as novel chemotherapeutics has not been available on class 2 HDACs so far. Here we present the structure of the bacterial FB188 HDAH (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes strain FB188) that reveals high sequential and functional homology to human class 2 HDACs. FB188 HDAH is capable to remove the acetyl moiety from acetylated histones. Several HDAC-specific inhibitors, which have been shown to inhibit tumor activity in both pre-clinical models and in clinical trials, also inhibit FB188 HDAH. We have determined the crystal structure of FB188 HDAH at a resolution of 1.6 angstroms in complex with the reaction product acetate, as well as in complex with the inhibitors suberoylanilide hydroxamic acid (SAHA) and cyclopentyle-propionyle hydroxamic acid (CypX) at a resolution of 1.57 angstroms and 1.75 angstroms, respectively. FB188 HDAH exhibits the canonical fold of class 1 HDACs and contains a catalytic zinc ion. The highest structural diversity compared to class 1 enzymes is found in loop regions especially in the area around the entrance of the active site, indicating significant differences among the acetylated proteins binding to class 1 and 2 HDACs, respectively.
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PMID:Crystal structure of a bacterial class 2 histone deacetylase homologue. 1624 51

Folate receptor (FR) has been proposed as a promising target for tumor drug targeting. The aim of this study was to increase the chemo-sensitivity of FR-positive cells to doxorubicin by folate-directed enzyme prodrug therapy (FDEPT). Folate conjugated penicillin-G amidase was prepared and its ability to hydrolyze N-(phenylacetyl) doxorubicin was measured by HPLC. Fluorescence and confocal image analysis revealed that Folate-PGA can be specifically delivered into FR-positive HeLa and SKOV3 tumor cells. In vitro cytotoxity assays, IC50 was reduced with N-(phenylacetyl) doxorubicin versus doxorubicin for HeLa (3.1-fold reduction; p<0.001) and SKOV3 (3.3-fold reduction; p<0.001) when Folate-PGA was specifically bound to the cells. Complete activation was confirmed in HeLa and SKOV3 cells pretreated with free folic acid (1 mM), where the combination of N-(phenylacetyl) doxorubicin with Folate-PGA did not show any significant cell toxicity to the IC50 of doxorubicin. Pharmacokinetic clearance and biodistribution studies in vivo showed that 125I-Folate-PGA was cleared from blood within 24 h and had significantly higher tumor uptake compared to 125I-PGA (p<0.05). These results demonstrate that the FDEPT approach may be a potential promising strategy to improve chemotherapy-resistant cancers therapeutic ratio and warranted future studies.
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PMID:Increase of doxorubicin sensitivity for folate receptor positive cells when given as the prodrug N-(phenylacetyl) doxorubicin in combination with folate-conjugated PGA. 1688 59

Infection with Helicobacter pylori (H. pylori) is a risk factor for the development of gastric cancer. Here we show that infection of gastric epithelial cells with 'cag' pathogenicity island (cagPAI)-positive H. pylori induced aberrant expression of activation-induced cytidine deaminase (AID), a member of the cytidine-deaminase family that acts as a DNA- and RNA-editing enzyme, via the IkappaB kinase-dependent nuclear factor-kappaB activation pathway. H. pylori-mediated upregulation of AID resulted in the accumulation of nucleotide alterations in the TP53 tumor suppressor gene in gastric cells in vitro. Our findings provide evidence that aberrant AID expression caused by H. pylori infection might be a mechanism of mutation accumulation in the gastric mucosa during H. pylori-associated gastric carcinogenesis.
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PMID:Helicobacter pylori infection triggers aberrant expression of activation-induced cytidine deaminase in gastric epithelium. 1741 70

Nasopharyngeal carcinoma (NPC) is one of the common malignant tumors in China. Radiotherapy and chemotherapy are the main therapy methods for NPC. To enhance the specific antitumor effect, a novel vector with radiosensitivity and tumor specificity was constructed in this study, which enables the reduction of dosage of radiation and chemotherapeutic drugs due to its double killing effect. Four DNA elements, Egr-1 promoter, Cytosine deaminase (CD) gene, hTERT promoter, Survivin antisense oligonucleotides were amplified and constructed in pcDNA3.1 vector. CD and Survivin gene expression in CNE-2 cells were detected by RT-PCR. High performance liquid chromatography (HPLC) was employed to determine the transformation from the prodrugs 5-FC to 5-FU. Hoechst33258 staining of the nuclei and methylthiazolyl tetrazolium(MTT) assay were applied to detect apoptosis and cell survivability, respectively. In addition, the anti-tumor effects were examined in vivo by injecting cells with different vectors into nude mice. Our results revealed a notable killing effect of combined treatment with 5-FC and radiation on CNE-2 cells transfected with vectors in vitro. This effect was especially notable on pEC-TS transferred cells, which showed 57% of cells were killed. In vivo, an obvious suppression of tumor was displayed in pEC-TS group, which was significantly different from other groups (p < 0.05). Consequently, this expression cassette may have a great therapeutic potential for the treatment of NPC.
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PMID:Role of enhanced radiosensitivity and the tumor-specific suicide gene vector in gene therapy of nasopharyngeal carcinoma. 1746 96

We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2'-deoxyzebularine 5'-triphosphate (dZTP). Because 2'-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine 5'-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent, and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.
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PMID:Activation of p16 gene silenced by DNA methylation in cancer cells by phosphoramidate derivatives of 2'-deoxyzebularine. 1900 82


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