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Cloning, construction of prokaryotic expression vector and expression ofEscherichia coli cytosine deaminase gene
Shengjun Ren,Huiqiu Jiang,Jianren Gu
Chinese Science Bulletin , 1998, DOI: 10.1007/BF02898917
Abstract: Cytosine deaminase gene ofEscherichia coli strain H-30 was cloned, and its initiation codon of ‘GTG’ was mutated to ‘ATG’ by PCR. Prokaryotic recombinant expression vector pBV220-CD was constructed. Clone with high enzyme activity were selected by detecting their specific activity of cytosine deaminase. 5-FC(5-FC, 5-fluorocytosine) could induce the lethal toxicity to cells containing active CD gene. DNA sequence analysis indicated that there were 16 altered bases and 5 of them resulted in the alteration of amino acids in predicted peptide by comparing DNA sequence of the clone H-30-CD-11 with high enzyme activity with CD gene reported in Gene Bank.
Cloning, construction of prokaryotic expression vector and expression of Escherichia coli cytosine deaminase gene

Shengjun Ren,Huiqiu Jiang,Jianren Gu,

科学通报(英文版) , 1998,
Abstract: Cytosine deaminase gene ofEscherichia coli strain H-30 was cloned, and its initiation codon of ‘GTG’ was mutated to ‘ATG’ by PCR. Prokaryotic recombinant expression vector pBV220-CD was constructed. Clone with high enzyme activity were selected by detecting their specific activity of cytosine deaminase. 5-FC(5-FC, 5-fluorocytosine) could induce the lethal toxicity to cells containing active CD gene. DNA sequence analysis indicated that there were 16 altered bases and 5 of them resulted in the alteration of amino acids in predicted peptide by comparing DNA sequence of the clone H-30-CD-11 with high enzyme activity with CD gene reported in Gene Bank.
AID/APOBEC cytosine deaminase induces genome-wide kataegis
Lada Artem G,Dhar Alok,Boissy Robert J,Hirano Masayuki
Biology Direct , 2012, DOI: 10.1186/1745-6150-7-47
Abstract: Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm), are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events. Reviewers This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.
Antitumor activity of mutant bacterial cytosine deaminase gene for colon cancer  [cached]
Long-Ying Deng,Jian-Ping Wang,Zhi-Fu Gui,Li-Zong Shen
World Journal of Gastroenterology , 2011, DOI: 10.3748/wjg.v17.i24.2958
Abstract: AIM: To evaluate bacterial cytosine deaminase (bCD) mutant D314A and 5-fluorocytosine (5-FC) for treatment of colon cancer in a mouse model. METHODS: Recombinant lentivirus vectors that contained wild-type bCD gene (bCDwt), and bCD mutant D314A gene (bCD-D314A) with green fluorescence protein gene were constructed and used to infect human colon carcinoma LoVo cells, to generate stable transfected cells, LoVo/null, LoVo/bCDwt or LoVo/bCD-D314A. These were injected subcutaneously into Balb/c nude mice to establish xenograft models. Two weeks post-LoVo cell inoculation, PBS or 5-FC (500 mg/kg) was administered by intraperitoneal (i.p.) injection once daily for 14 d. On the day after LoVo cell injection, mice were monitored daily for tumor volume and survival. RESULTS: Sequence analyses confirmed the construction of recombinant lentiviral plasmids that contained bCDwt or bCD-D314A. The lentiviral vector had high efficacy for gene delivery, and RT-PCR showed that bCDwt or bCD-D314A gene was transferred to LoVo cells. Among these treatment groups, gene delivery or 5-FC administration alone had no effect on tumor growth. However, bCDwt/5-FC or bCD-D314A/5-FC treatment inhibited tumor growth and prolonged survival of mice significantly (P < 0.05). Importantly, the tumor volume in the bCD-D314A/5-FC-treated group was lower than that in the bCDwt/5-FC group (P < 0.05), and bCD-D314A plus 5-FC significantly prolonged survival of mice in comparison with bCDwt plus 5-FC (P < 0.05). CONCLUSION: The bCD mutant D314A enhanced significantly antitumor activity in human colon cancer xenograft models, which provides a promising approach for human colon carcinoma therapy.
Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast
Alessandra Mallano, Silvia Zamboni, Giulia Carpinelli, Filippo Santoro, Michela Flego, Alessandro Ascione, Mara Gellini, Marina Tombesi, Franca Podo, Maurizio Cianfriglia
BMC Biotechnology , 2008, DOI: 10.1186/1472-6750-8-68
Abstract: An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU.The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.The ability of cytosine deaminase (CD) to convert the clinically used antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer agent such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to design innovative anticancer therapies [1,2]. Therefore, CD-based enzyme/prodrug strategies are under investigation to model gene or antibody directed enzyme-prodrug therapy (GDEPT/ADEPT) for achieving high local concentration of 5-FU without significant systemic toxicity [3,4]. In in vivo animal model, the CD gene/enzyme which is not naturally expressed in mammals are first introduced into the cells of a tumour by specific antibodies [5-7], modified microorganisms such as bacteria and viruses or synthetic vectors (reviewed by Springer et al., 2007)[4]. When the discrimination between tumor and normal tissue enzyme levels is sufficient, 5-FC is given i.v., which is converted into 5-FU by CD within the tumor [8]. A convincing demonstration that such a complex system can be developed for
Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase  [PDF]
Artem G. Lada,Elena I. Stepchenkova,Irina S. R. Waisertreiger,Vladimir N. Noskov,Alok Dhar,James D. Eudy,Robert J. Boissy,Masayuki Hirano,Igor B. Rogozin,Youri I. Pavlov
PLOS Genetics , 2013, DOI: 10.1371/journal.pgen.1003736
Abstract: Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis.
Adenovirus-mediated interleukin-12 gene transfer combined with cytosine deaminase followed by 5-fluorocytosine treatment exerts potent antitumor activity in Renca tumor-bearing mice
Kyung-Sun Hwang, Won-Kyung Cho, Jinsang Yoo, Hwan-Jung Yun, Samyong Kim, Dong-Soo Im
BMC Cancer , 2005, DOI: 10.1186/1471-2407-5-51
Abstract: Adenoviral vectors were constructed encoding one or both subunits of murine IL-12 (Ad.p35, Ad.p40 and Ad.IL-12) or cytosine deaminase (Ad.CD). The functionality of the IL-12 or CD gene products expressed from these vectors was validated by splenic interferon (IFN)-γ production or viability assays in cultured cells. Ad.p35 plus Ad.p40, or Ad.IL-12, with or without Ad.CD, were administered (single-dose) intratumorally to Renca tumor-bearing mice. The animals injected with Ad.CD also received 5-FC intraperitoneally. The antitumor effects were then evaluated by measuring tumor regression, mean animal survival time, splenic natural killer (NK) cell activity and IFN-γ production.The inhibition of tumor growth in mice treated with Ad.p35 plus Ad.p40 and Ad.CD, followed by injection of 5-FC, was significantly greater than that in mice treated with Ad.CD/5-FC, a mixture of Ad.p35 plus Ad.p40, or Ad.GFP (control). The combined gene transfer increased splenic NK cell activity and IFN-γ production by splenocytes. Ad.CD/5-FC treatment significantly increased the antitumor effect of Ad.IL-12 in terms of tumor growth inhibition and mean animal survival time.The results suggest that adenovirus-mediated IL-12 gene transfer combined with Ad.CD followed by 5-FC treatment may be useful for treating cancers.Gene therapy for treating cancers has been intensively investigated. Therapeutic gene transfer using viral or nonviral vectors has been shown to be clinically feasible and safe. However, the therapeutic outcome appears not to be high as expected, presumably because the in vivo gene delivery efficiency is low. In this sense, a more effective modality is needed to improve the therapeutic benefit. Combined transfer of genes with different mechanisms of action may potentate the therapeutic outcome. Indeed, combination cancer gene therapy using cytokine and suicide genes has been shown to be more effective than single gene transfer in animal models [1-9].Interleukin (IL)-12 is a heterodim
Purification and characterization of Aspergillus parasiticus cytosine deaminase for possible deployment in suicide gene therapy  [PDF]
Hassan Zanna, A. J. Nok, S. Ibrahim, H. M. Inuwa
Advances in Biological Chemistry (ABC) , 2012, DOI: 10.4236/abc.2012.22018
Abstract: Cytosine Deaminase (CD) from Aspergillus parasiticus was purified and characterized. Time course for maximal CD production (50 μmol/min/mg) was at 72 hrs. The enzyme was purified 387.73 folds with an overall yield of 13%. The CD had pH optimum of 7.2, a temperature optimum of 40℃ - 45℃, activation of energy (Ea) of 8.4 KJ/mol and a t1/2 of 1.10 hr. A. parasiticus CD stoichiometrically deaminated Cyto-sine and 5-fluorocytosine (5-FC) with the KM values of 0.19mM and 0.30 mM respectively. Studies on the effect of pH on KM and Vmax gave pKa1 of 5.8 and pKa2 of 6.3 with enthalpy of ionization of 43.01 KJ/mole suggesting histidine in the active site. The enzyme was strongly inhibited by Ca2+, Co2+, Zn2+ and Hg2+ and completely inhibited by Cu2+ and Fe2+ at 50 mM. Therefore, A. parasiticus CD can be compared with CDs from other sources that are used in suicide gene therapy.
Calcitriol and TO-901317 Interact in Human Prostate Cancer LNCaP Cells
Jing-Huan Wang,Pentti Tuohimaa
Gene Regulation and Systems Biology , 2008,
Abstract: Vitamin D receptor (VDR) and liver X receptor (LXR) are nuclear receptors, which regulate gene transcription upon binding of their specific ligands. VDR seems to play a role in the regulation of prostate cancer cell proliferation. ATPbinding cassette transporter A1 (ABCA1) is known to be a target gene of LXR and it has been reported to be inhibited by androgen and to be involved in the regulation of LNCaP proliferation. We fi nd that calcitriol (1α,25(OH)2D3) inhibits both basal and a LXR agonist, TO-901317, induced ABCA1 mRNA expression but has no effect on the mRNA expression of ATP-binding cassette transporter G1 (ABCG1), LXRα nor LXRβ. TO-901317 increases both basal and calcitriol induced 25-hydroxyvitamin D3-24-hydroxylase (CYP24) mRNA expression and it slightly but significantly inhibits VDR mRNA expression. The inhibition of ABCA1 by calcitriol appears to be androgen-independent. Cell growth assay shows that when each of calcitriol and 5α-dihydrotestosterone (DHT) was co-treated with ABCA1 blocker, glybenclamide, cell-growth is significantly decreased compared to their own treatments respectively. Our study suggests a possible interaction between calcitriol and TO-901317 in LNCaP cells. Alike DHT, the inhibition of ABCA1 by calcitriol may be involved in its regulation of LNCaP growth.
Synthesis of Pregnane Derivatives, Their Cytotoxicity on LNCap and PC-3 Cells, and Screening on 5α-Reductase Inhibitory Activity  [PDF]
Sujeong Kim,Eunsook Ma
Molecules , 2009, DOI: 10.3390/molecules14114655
Abstract: A series of epoxy- and/or 20-oxime pregnanes were synthesized from commercially available pregnenolone. Compounds 1, 3, 7, 8 and 11-13 were evaluated for cytotoxicity activity towards LNCaP (androgen-dependent) and PC-3 (androgenindependent) prostate cancer cells. Compound 13 showed the highest activity on both LNCaP (IC50 15.17 μM) and PC-3 (IC50 11.83 μM) cell lines. Compound 11 showed weak activity on LNCaP cells (IC50 71.85 μM) and 8 showed the weak activity on PC-3 cells (IC50 68.95 μM), respectively. The 5α-reductase II (5AR2) inhibitory effects of compounds 1-3, 5 and 7-13 were investigated in a convenient screening model, in which compounds 5, 8, 11 and 12 were observed to be potential inhibitors of 5α-reductase, in particular, the 4-azasteroid 11, that also inhibited cell proliferation of androgen-dependent cells and 8, that in addition inhibited PC-3 cells more potently than LNCaP cells.
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