• Journal of the Korean Chemical Society
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• v.32 no.4
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• pp.333-341
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• 1988

The bio-electrode for cytosine has been constructed by immobilizing Proteus mirabilis and Citrobacter freundii on an ammonia gas-sensor. Bacteria containing cytosine deaminase convert one molecule of cytosine into one molecule of ammonia. The Proteus mirabilis bacterial electrode showed linear response to cytosine concentration in the $1.0{\times}10^{-3}\;-\;5.0{\times}10^{-2}$M with a slope of 45-48 mV/decade in 0.2 M phospbate buffer solution at pH 8.4. The Citrobacter freundii bacterial electrode showed linear response to cytosine concentration in the $7.0{\times}10^{-5}\;-\;7.0{\times}10^{-3}$M with a slope of 48 mV/decade in 0.05M phosphate buffer solution at pH 7.6. These electrode were investigated for the effects of pH, temperature, buffer solutions, amounts of bacteria, interferences, inorganic salts and lifetime.

• Biomolecules & Therapeutics
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• v.13 no.3
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• pp.143-149
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• 2005

We have demonstrated previously on a replication incompetent recombinant adenoviral vector, AdLPCD, in which the expression of cytosine deaminase (CD) gene is driven by the tumor-specific L-plastin promoter. The object of this study was to evaluate the efficacy of AdLPCD together with 5-fluorocytosine (5-FC) in suppression of the growth of established human tumor cells of ovary, Consistent with the knowledge that infection of OVCAR-3 cells with AdLPCD resulted in expression of a functional intracellular CD enzyme capable of converting 5-FC to 5-fluorouracil (5-FU) (Chung and Deisseroth, 2004), statistically significant differences in cytotoxicity were observed when AdLPCD infected cells were also exposed to 5-FC for 6 days (p=0.05), 9 days (p<0.0005) and 12 days (p<0.005), compared to 5-FC exposure alone, These results indicate that the CD gene delivered by adenoviral vector could efficiently sensitize OVCAR-3, otherwise non-toxic 5-FC. On the other hand, SKOV-3 cells, an ovarian carcinoma cell line, were more resistant to the CD/5-FC strategy compared with OVCAR-3 cells under the same condition. The results of present study suggest that the replacement of 5-FU with CD/5-FC in combination chemotherapy would be less toxic and much greater cytotoxicity than the conventional combination chemotherapy in some patients.

• Archives of Pharmacal Research
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• v.27 no.6
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• pp.633-639
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• 2004

The expression of therapeutic transgenes in recombinant adenoviral vectors is a major cause of toxicity in dividing cancer cells as well as non dividing normal cells. To solve the problem of toxicity to normal cells, we have reported on a recombinant adenoviral vector system （AdLP-） in which the expression of the transgene is directed by the tumor-specific L-plastin promoter （LP） （Chung et al., 1999）. The object of this study was to generate a recombinant adenoviral vector system which would generate tumor cell specific expression of cytosine deaminase （CD） gene. We report the construction of a replication-incompetent adenoviral vector in which CD is driven by the L-plastin promoter （AdLPCD）. Infection of 293 cells by AdLPCD generated the functional CD protein as measured by HPLC analysis for the conversion of 5-Fluorocy-tosine （5-FC） to 5-Fluorouracil （5-FU）. HPLC analysis in conjunction with counting radioactivity for ［6-$^3$H］-5FC and ［6-$^3$H］-5FU demonstrated vector dose-dependent conversion of 5-FC to 5-FU in AdLPCD infected ovarian cancer cells. The results from present and previous studies（Peng et al., 2001； Akbulut et al., 2003） suggest that the use of the AdLPCD／5-FC system may be of value in the treatment of cancer including microscopic ovarian cancer in the peritoneal cavity.

• Biomolecules & Therapeutics
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• v.17 no.2
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• pp.138-143
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• 2009

We have reported previously on a replication incompetent recombinant adenoviral vector, AdLPCD, in which the expression of cytosine deaminase gene (CD) is driven by the tumor-specific L-plastin promoter. AdLPCD vector had been evaluated for its efficacy of chemosensitization of ovarian cancer cells to 5-FC. In spite of the fact that ovarian cancer cells, i.e., OVCAR-3 and SK-OV-3, are capable for adenoviral transduction judged by LacZ reporter gene analysis, two cell lines demonstrated quite different sensitivities toward AdLPCD/5-FC system. In OVCAR-3 cells, infection of AdLPCD followed by exposure to 5-FC resulted in the suppression of cell growth with statistical significance. On the other hand, SK-OV-3 cells were more resistant to the CD/5-FC strategy compared with OVCAR-3 cells under the same condition. The object of study was to investigate factors that would determine the sensitivity to AdLPCD/5-FC. We evaluated conversion rate of 5-FC to 5-FU after infection of AdLPCD by HPLC analysis, $IC_{50}$ of 5-FU, the expression level of integrin receptors i.e., ${\alpha}v{\beta}3$ and ${\alpha}v{\beta}5$, and status of p53 in OVCAR-3 and SK-OV-3 cells. The results indicated that OVCAR-3 cells have few favorable features compared with SK-OV-3 cells to be more effective to the AdLPCD/5-FC strategy; higher level of ${\alpha}v{\beta}5$ integrin, higher rate of conversion of 5-FC into 5-FC, and lower $IC_{50}$ of 5-FU. The results suggest that the replacement of 5-FU with CD/5-FC in combination chemotherapy would be less toxic and much greater cytotoxicity than the conventional combination chemotherapy in some patients.

• Journal of Korean Neurosurgical Society
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• v.30 no.sup1
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• pp.13-19
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• 2001

Objective : We investigated the feasibility of a double suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type 1 thymidine kinase(TK) gene and the Escherichia coli cytosine deaminase(CD) gene, via a recombinant adenoviral vector and ganciclovir(GCV) and/or 5-fluorocytosine(5-FC) treatment, in C6 glioma cells. Methods : Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the ${\beta}$-galactosidase gene and then staining cells with 5-bromo-4-chloro-3-indolyl-13-D-galactoside. CD/TK expression in cells infected with adenovirus bearing the CD/TK gene(ad-CD/TK) was examined by immunoblotting analysis. For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-${\Delta}E1$(as a control). After addition of a variety of concentrations of GCV and 5-FU, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection. Result : C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more. In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK. Obvious cytotoxicity(>50% inhibition) was observed in the presence of 5-FC at concentrations greater than 30ug/ml or GCV at concentrations greater than 0.3ug/ml at a multiplicity of infection of 100. Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after sinlge-prodrug treatments, indicating additive effects of the prodrug treatments. Conclusion : The administration of a double-suicide gene/prodrug therapy might have great potential in the treatment of brain tumors.

• Korean Journal of Agricultural Science
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• v.46 no.4
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• pp.885-895
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• 2019

Plant biotechnologists have long dreamed of technologies to manipulate genes in plants at will. This dream has come true partly through the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, which now has been used to edit genes in several important crops. However, there are many restrictions in editing a gene precisely using the CRISPR/Cas9 technology because CRISPR/Cas9 may cause deletions or additions in some regions of the target gene. Several other technologies have been developed for gene targeting and precision editing. Among these, base editors might be the most practically and efficiently used compared to others. Base editors are tools which are able to cause a transition from cytosine into thymine, or from adenine into guanine very precisely on specific sequences. Cytosine base editors basically consist of nCas9, cytosine deaminase, and uracil DNA glycosylase inhibitor (UGI). Adenine base editors consist of nCas9 and adenine deaminase. These were first developed for human cells and have since also been applied successfully to crops. Base editors have been successfully applied for productivity improvement, fortification and herbicide resistance of crops. Thus, base editor technologies start to open a new era for precision gene editing or breeding in crops and might result in revolutionary changes in crop breeding and biotechnology.

• Biomolecules & Therapeutics
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• v.14 no.3
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• pp.148-151
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• 2006

We have previously reported that 2.4 kb of L-plastin promoter (LP) could regulate the expression of adenoviral vector (AV) exogenous genes in a tumor cell specific manner. In the present study, we tested if the replication competent AdLPE1A vector results in a direct cytotoxic effect in hepatocelluar carcinoma (HCC) cells. In vitro cytotoxicity tests were carried out with replication-competent (AdLPE1A) and -incompetent (AdLPCD) LP-driven vectors. AdLPE1A is an AV in which LP was inserted 5' to the E1A and E1B genes. The AdLPCD vector contains LP and the E. coli cytosine deaminase (CD) gene in transcription unit. Exposure of cells to AdLPE1A generated a significant cytotoxic effect as compared to the control. Almost 90% of the cell had manifested the characteristic cytopatic effect on day 9 after infection of cells with 10 MOI of AdLPE1A. On the other hand, almost 35% of the cells were left when the cells had been treated with 100 MOI of AdLPCD together with 5-FC on day 9 when compared with the cells which had never been exposed neither 5-FC nor AdLPCD. These results showed that the replication competent AdLPE1A vector could kill the HepG2 cells directly by the oncolytic effect of the virus. The replication competent AV vector carrying viral E1A generated greater cytotoxic effect than the replication incompetent AV, which contains the CD prodrug activation transcription unit without E1A, in HepG2 cells.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7489-7496
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• 2013

This study was conducted to investigate enhancement of anti-tumor effects of the lentogenic Newcastle disease virus Clone30 strain (NDV rClone30) expressing cytosine deaminase (CD) gene against tumor cells and in murine groin tumor-bearing models. Cytotoxic effects of the rClone30-CD/5-FC on the HepG2 cell line were examined by an MTT method. Anti-tumor activity of rClone30-CD/5-FC was examined in H22 tumor-bearing mice. Compared to the rClone30-CD virus treatment alone, NDV rClone30-CD/5-FC at 0.1 and 1 MOIs exerted significant cytotoxic effects (P<0.05) on HepG2 cells. For treatment of H22 tumor-bearing mice, recombinant NDV was injected together with 5-FC given by either intra-tumor injection or tail vein injection. When 5-FC was administered by intra-tumor injection, survival for the rClone30-CD/5-FC-treated mice was 4/6 for 80 days period vs 1/6, 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC and saline alone, respectively. When 5-FC was given by tail vein injection, survival for the rClone30-CD/5-FC-treated mice was 3/6 vs 2/6, 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC or saline alone, respectively. In this study, NDV was used for the first time to deliver the suicide gene for cancer therapy. Incorporation of the CD gene in the lentogenic NDV genome together with 5-FC significantly enhances cell death of HepG2 tumor cells in vitro, decreases tumor volume and increases survival of H22 tumor-bearing mice in vivo.

• International Journal of Stem Cells
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• v.16 no.4
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• pp.438-447
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• 2023

Recently, ex-vivo gene therapy has emerged as a promising approach to enhance the therapeutic potential of mesenchymal stem cells (MSCs) by introducing functional genes in vitro. Here, we explored the need of using selection markers to increase the gene delivery efficiency and evaluated the potential risks associated with their use in the manufacturing process. We used MSCs/CD that carry the cytosine deaminase gene (CD) as a therapeutic gene and a puromycin resistance gene (PuroR) as a selection marker. We evaluated the correlation between the therapeutic efficacy and the purity of therapeutic MSCs/CD by examining their anti-cancer effect on co-cultured U87/GFP cells. To simulate in vivo horizontal transfer of the PuroR gene in vivo, we generated a puromycin-resistant E. coli (E. coli/PuroR) by introducing the PuroR gene and assessed its responsiveness to various antibiotics. We found that the anti-cancer effect of MSCs/CD was directly proportional to their purity, suggesting the crucial role of the PuroR gene in eliminating impure unmodified MSCs and enhancing the purity of MSCs/CD during the manufacturing process. Additionally, we found that clinically available antibiotics were effective in inhibiting the growth of hypothetical microorganism, E. coli/PuroR. In summary, our study highlights the potential benefits of using the PuroR gene as a selection marker to enhance the purity and efficacy of therapeutic cells in MSC-based gene therapy. Furthermore, our study suggests that the potential risk of horizontal transfer of antibiotics resistance genes in vivo can be effectively managed by clinically available antibiotics.

• Nuclear Medicine and Molecular Imaging
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• v.40 no.5
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• pp.237-242
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• 2006

Knowledge of molecular mechanisms governing malignant transformation brings new opportunities for therapeutic intervention against cancer using novel approaches. One of them is gene therapy based on the transfer of genetic material to an organism with the aim of correcting a disease. The application of gene therapy to the cancer treatment has led to the development of new experimental approaches such as suicidal gene therapy, inhibition of oncogenes and restoration of tumor-suppressor genes. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a prodrug into a toxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1-tk) and cytosine deaminase (CD). Especially, physicians and scientists of nuclear medicine field take an interest In suicidal gene therapy because they can monitor the location and magnitude, and duration of expression of HSV1-tk and CD by PET scanner.