CRISPR-mediated base editing in mice using cytosine deaminase base editor 4

BACKGROUND: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). RESULTS: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. CONCLUSIONS: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.

A Potential Therapy Using Engineered Stem Cells Prevented Malignant Melanoma in Cellular and Xenograft Mouse Models / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: In the present study, human neural stem cells (hNSCs) with tumor-tropic behavior were used as drug delivery vehicle to selectively target melanoma. A hNSC line (HB1.F3) was transduced into two types: one expressed only the cytosine deaminase (CD) gene (HB1.F3. CD) and the other expressed both CD and human interferon-β (IFN-β) genes (HB1.F3.CD. IFN-β). MATERIALS AND METHODS: This study verified the tumor-tropic migratory competence of engineered hNSCs on melanoma (A375SM) using a modified Boyden chamber assay in vitro and CM-DiI staining in vivo. The antitumor effect of HB1.F3.CD and HB1.F3.CD.IFN-β on melanoma was also confirmed using an MTT assay in vitro and xenograft mouse models. RESULTS: A secreted form of IFN-β from the HB1.F3.CD.IFN-β cells modified the epithelial-mesenchymal transition (EMT) process and metastasis of melanoma. 5-Fluorouracil treatment also accelerated the expression of the pro-apoptotic protein BAX and decelerated the expression of the anti-apoptotic protein Bcl-xL on melanoma cell line. CONCLUSION: Our results illustrate that engineered hNSCs prevented malignant melanoma cells from proliferating in the presence of the prodrug, and the form that secreted IFN-β intervened in the EMT process and melanoma metastasis. Hence, neural stem cell-directed enzyme/prodrug therapy is a plausible treatment for malignant melanoma.

Apoptosis induced by transferring cytosine deaminase suicide gene into kinds of cancer cells with novel polyamidoamine dendrimers / 药学学报

This study demonstrates efficacy of a novel polyamidoamine dendrimers (PAMAM dendrimers) with pentaerythritol derivatives as the core (G5 PD dendrimer) in deliver of the cytosine deaminase (CD) gene and EGFP gene fusion plasmid into different tumor cell lines to induce apoptosis. The physical and chemical properties of G5 PD dendrimers in terms of DNA complexation, particulate properties and transfection efficiencies were investigated and compared with commercial gene vectors PEI 25 kDa. The optimum ratio of G5 PD dendrimer complexed with plasmid DNA was 0.2:1, and the particle size of the complex was (100±5) nm. Compared with the commercial gene carriers PEI, G5 PD dendrimer exhibited a higher transfection efficiency at the weight ratio of 1:1 in three different cell lines, given the fact that PEI are different from PAMAM dendrimers in terms of molecular structure. Furthermore, the cytotoxicity assays of the cell lines transfected with G5 PD dendrimer/pCD-EGFP-N1 followed by exposure to various concentrations of 5-fluorocytosine (5-FC) also showed that the transfected cell lines could generate a very low amount of 5-FC to 5-fluorouracil (5-FU) in a short period of time, which indicating the high expression level of CD gene in the cell line. These results indicate that the CD/5FC system of G5 PD dendrimer has an excellent efficacy in gene delivery.

Effect of endothelial progenitor cells modified by suicide gene on growth of hepatocellar carcinoma in orthotopic tumor-transplanted mice / 肿瘤

Objective: To investigate the suppression effect of mouse marrow-derived endothelial progenitor cells (EPCs) modified by cytosine deaminase (CD) gene and enzyme prodrug 5-fluorocytosine (5-Fc) on the growth of hepatocellar carcinoma in orthotopic H22 cell-transplanted mice. Methods: The EPCs were transfected by lentiviral vector carrying CD gene (plenti6.3-EGFP-CD) using Polybrene technique. The mouse hepatoma H22 cells were treated by the supernatant of EPCs carrying CD gene and 5-Fc, and then the number and morphology of the cells were observed under an inverted microscope. C57BL/6 mice bearing orthotopic transplanted H22 hepatocellar carcinoma were treated by EPCs carrying CD gene and 5-Fc. The volume of tumor in mice was monitored by magnetic resonance imaging, and the apoptosis in tumor was tested by terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay. The expression of CD protein in the transplanted tumor was identified by Western blotting. Results: The proliferation of H22 cells treated by the supernatant of EPCs carrying CD gene combined with 5-Fc was significantly reduced. Compared with the control, the tumor growth in the hepatocellular carcinoma orthotopic-transplantation mouse models treated by EPCs carrying CD gene and 5-Fc was inhibited to (47.29±5.81)% (P < 0.05), and the apoptosis index of tumor cells was markedly increased [(39.98±5.13)%]. Conclusion: The EPCs modified by CD gene combined with 5-Fc administration can effectively inhibit the proliferation of the mouse orthotopic transplanted hepatocellular carcinoma, and induce the apoptosis of tumor cells. Copyright © 2014 by TUMOR.

Selective Delivery of a Therapeutic Gene for Treatment of Head and Neck Squamous Cell Carcinoma Using Human Neural Stem Cells

OBJECTIVES: Based on studies of the extensive tropism of neural stem cells (NSCs) toward malignant brain tumor, we hypothesized that NSCs could also target head and neck squamous cell carcinoma (HNSCC) and could be used as a cellular therapeutic delivery system. METHODS: To apply this strategy to the treatment of HNSCC, we used a human NSC line expressing cytosine deaminase (HB1.F3-CD), an enzyme that converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), an anticancer agent. HB1. F3-CD in combination with 5-FC were cocultured with the HNSCC (SNU-1041) to examine the cytotoxicity on target tumor cells in vitro. For in vivo studies, an HNSCC mouse model was created by subcutaneous implantation of human HNSCC cells into athymic nude mice. HB1.F3-CD cells were injected into mice using tumoral, peritumoral, or intravenous injections, followed by systemic 5-FC administration. RESULTS: In vitro, the HB1.F3-CD cells significantly inhibited the growth of an HNSCC cell line in the presence of the 5-FC. Independent of the method of injection, the HB1.F3-CD cells migrated to the HNSCC tumor, causing a significant reduction in tumor volume. In comparison to 5-FU administration, HB1.F3-CD cell injection followed by 5-FC administration reduced systemic toxicity, but achieved the same level of therapeutic efficacy. CONCLUSION: Transplantation of human NSCs that express the suicide enzyme cytosine deaminase combined with systemic administration of the prodrug 5-FC may be an effective regimen for the treatment of HNSCC.

Effects of tissue specific cytosine deaminase/5-fluorocytosine thermotherapy on hepatic metastasis of colonic carcinoma in nude mice / 中华消化外科杂志

Objective To investigate the effects of tissue specific cytosine deaminase/5-fluorocytosine (CD/5-FC) thermotherapy on hepatic metastasis of colonic carcinoma in nude mice. Methods Forty-five nude mice were randomly divided into control group, 5-FC group and 5-FC thermotherapy group according to the random number table (15 mice in each group). Mice models of hepatic metastasis of colonic carcinoma were established by portal vein injection of LoVo/CEACD cells. The hepatic metastasis rate and number of metastatic nodules of the 3 groups were compared by ehi-square test and one-way ANOVA. The pathological changes in tumor tissues and apoptotic index of tumor cells were observed. The expression of the CD gene in tumor tissues was detected by fluorescent quantitative RT-PCR and Western blot. Results The number of metastatic nodules and liver metas-tasis rate were 4.6±1.3 and 100.0% in control group, 2.2±1.0 and 60.0% in 5-FC group, 0.5±0.8 and 13.3% in 5-FC thermotherapy group, with statistical difference among the 3 groups (F=25.898, χ2=5.208, 19.548, 5.168, P<0.05). The mean apoptotic indexes of tumor cells of the 3 groups were 4.6%, 9.9% and 17.4%, respectively. Vacuolar degeneration, cell necrosis, cytolysis and apoptotic bodies were mostly observed in the 5-FC thermotherapy group. The expression of CD gene in tumor tissue was detected in all the groups. Conclusion Tissue specific CD/5-FC thermotherapy has inhibitory effects on the hepatic metastasis of LoVo cells transfected with CD gene.

Inhibitory effect of mutant cytosine deaminase D314A against human colon cancer cells / 中国肿瘤生物治疗杂志

Objective:To construct a mutant D314A of Escherichia coli cytosine deaminase (EC-CD, substitution of an alanine (A) for the aspartic acid (D) at position 314 of cytosine deaminase) and investigate its antitumor effect. Methods: Eukaryotic expression plasmid containing EC-CD gene (pcDNA3.1-CD~(wt)) was constructed, and the mutant pcDNA3.1-CD~(D314A) plasmid, with aspartic acid (D) at position 314 of EC-CD gene substituted by alanine (A) (EC-CD~(D314A)), was established by site-directed mutation. EC-CD~(wt) and EC-CD~(D314A) were transfected into human colon cancer cell line LoVo via Lipofectamine~(tm) 2000, and positive LoVo-CD~(wt) and LoVo-CD~(D314A) cells stably expressing corresponding genes were selected by G418. The cytotoxicity and bystander effects of EC-CD and EC-CD~(D314A) genes on LoVo cells were e-valuated by MTT assay. Results: The mutant D314A was confirmed by sequence analysis. EC-CD and EC-CD~(D314A) mRNA were expressed after transfected into LoVo cells. The IC_(50) of Lovo-CD~(D314A) cells was (85.13±0.60) mmol/L, which was significantly lower than that of LoVo-CD~(wt) cells ([689.76±0.45] μmol/L, P=0.000). Bystander effect assay showed that, when at the ratio of 30%, the survival rates of LoVo-CD~(wt) cells and Lovo-CD~(D314A) cells were (48.5±0.49)% and (17.3±0.40) % (P = 0.000), respectively. Conclusion: Mutatant EC-CD gene (EC-CD~(D314A)) has a significantly in-creased antitumor effect on LoVo cells compared with wild type EG-CD gene, and it may become a new candidate gene for tumor gene therapy.

Cell-killing effects of adenovirus-mediated transfer of CD :: UPP gene directed by mdr1 promoter on Taxol-resistant ovarian cancer cells / 肿瘤

Objective: To evaluate the specific killing effects of the adenovirus-mediated transfer of cytosine deaminase gene and uracil phosphoribosyltransferase gene (CD::UPP) directed by the mdrl promoter combined with 5-fluorocytosine (5-FC) on Taxol-resistant ovarian cancer cells in vitro and in vivo. Methods: The adenovirus vector carrying the mdrl-CD :: UPP gene was amplified, purified, and transferred into Taxol-resistant A2780/Taxol ovarian cancer cells and A2780 cells. The expression of mdrl and CD :: UPP was detected by RT-PCR. After addition of 5-FC, the cell proliferation was assessed by MTT assay. The female BALB/c nude mice bearing tumor xenografts were injected adenovirus vector containing mdrl-CD :: UPP gene and then administered 5-FC. The growth of transplanted tumors was observed. Results: Mdrl and CD :: UPP gene were stably expressed in A2780/Taxol cells. The growth speed of A2780/Taxol cells was significantly slower than A2780 cells after transfection. Transfection of CD :: UPP gene combined with administration of 5-FC induced bystander effect of A2780/ Taxol cells on neighbor cells. The tumor growth in test group was significantly inhibited compared with control group (569.10 ± 187.93) mm3 vs (2 111.98 ± 230.82) mm3, P < 0.01. Conclusion: The mdrl promoter in an adenovirus vector regulates the specific expression of CD :: UPP in A2780/Taxol cells. This system specifically kills Taxol-resistant ovarian cancer cells.

The Transfection of Cytosine Deaminase Gene and the Cell Killing Effects of Administration of 5-Fluorocytosine in Colon Cancer Cell Lines

PURPOSE: Among cancer gene therapies, the aims to eliminate malignant cells using genes as drugs as substitutes for conventional therapy, the use of bacterial cytosine deaminase (CD) which can convert the nontoxic 5-fluorocytosine (5-FC) to toxic 5-fluorouracil (5-FU), has been reported to provide a useful system for the selective killing of gene- modified mammalian tumor cells. Recently the transfection and expression of the CD gene, and the toxicity of 5-FC in eukaryotic cells, have been reported in colon, prostate and breast cancers, as well as in glioblastomas. To evaluate the growth inhibition effects in WiDr and LoVo colorectal cancer cells, after CD gene transfection and 5-FC administration, for the selective killing of cancer cells. METHODS: WiDr and LoVo colon cancer cell lines were cultured and the absorbencies for the percentage survival, on days 2, 4, 6, 8, and 10 of the culture, estimated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT) test. The experimental subjects were divided as follows; Group 1: administration of 5-FC only, Group 2: administration of 5-FU only, Group 3: administration of 5-FC after CD gene transfection using pCMV/CD-1 plasmid vector. Each experiment, on days 2, 4, 6, 8 and 10 of the culture after the administration of 5-FC or 5-FU concentration of 10, 100 and 1000 uM, was triplicated and calculated the mean with standard deviation. The bacterial CD gene was transfected into each cell lines, using the lipofectin method, and the transfection and expression of the CD protein identified with beta-galactosidase immunohistochemical staining and western blotting. The growth inhibition effects in group 3 were compared with those in group 1 and 2, and the bystander effects and cell recoveries, after finishing the 5-FC treatment, evaluated until day 20 of the culture. RESULTS: The calculated survival percentages by from the MTT tests in the WiDr and LoVo cells revealed 110~150% enhancing effects after the administration of 5-FC concentration of 10, 100 and 1000micro M (group 1), and 30~90% after the administration of 5-FU at the same concentrations (group 2), which were shown to be statistically significantly (P<0.001). After transfection of pCMV/CD-1 plasmid vector into the WiDr and LoVo cells using the lipofectin method, the transfection rate of the WiDr and LoVo cells were 4.2 +/- 0.6% and 13.8 +/- 0.8%, respectively. Also the CD protein, with the polyclonal anti-CD antibody, by western blot, revealed weak expression on day 2, followed by strong expression on day 4, which progressively decreased by days 6 and 8 in the WiDr cells. Conversely, the LoVo cells showed weak expression on day 2, which progressively increased by days 4 and 6, was followed by the strongest expression on day 8 in LoVo cells. About 60~85% significant growth inhibition effects (P<0.001) were revealed in group 3, with proportionally different effects, corresponding to the CD gene transfection rate, concentration of 5-FC and duration of the culture. The 60~85% growth inhibition effects were complex of the response to the transfection rate and the bystander effects of CD expressed cells to the CD non-expressed cells. CONCLUSION: From our results, the administration of 5-FC after CD gene transfection revealed statistically significant growth inhibition effects of 60~85%, which were complex of the effects of CD expressed cells and the bystander effects to the CD non-expressed cells, and proportionally corresponding to the transfection rate, the duration of the CD protein expression and the concentration of 5-FC.

The Transfection of Cytosine Deaminase Gene and the Cell Killing Effects of Administration of 5-Fluorocytosine in Colon Cancer Cell Lines

PURPOSE: Among cancer gene therapies, the aims to eliminate malignant cells using genes as drugs as substitutes for conventional therapy, the use of bacterial cytosine deaminase (CD) which can convert the nontoxic 5-fluorocytosine (5-FC) to toxic 5-fluorouracil (5-FU), has been reported to provide a useful system for the selective killing of gene- modified mammalian tumor cells. Recently the transfection and expression of the CD gene, and the toxicity of 5-FC in eukaryotic cells, have been reported in colon, prostate and breast cancers, as well as in glioblastomas. To evaluate the growth inhibition effects in WiDr and LoVo colorectal cancer cells, after CD gene transfection and 5-FC administration, for the selective killing of cancer cells. METHODS: WiDr and LoVo colon cancer cell lines were cultured and the absorbencies for the percentage survival, on days 2, 4, 6, 8, and 10 of the culture, estimated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT) test. The experimental subjects were divided as follows; Group 1: administration of 5-FC only, Group 2: administration of 5-FU only, Group 3: administration of 5-FC after CD gene transfection using pCMV/CD-1 plasmid vector. Each experiment, on days 2, 4, 6, 8 and 10 of the culture after the administration of 5-FC or 5-FU concentration of 10, 100 and 1000 uM, was triplicated and calculated the mean with standard deviation. The bacterial CD gene was transfected into each cell lines, using the lipofectin method, and the transfection and expression of the CD protein identified with beta-galactosidase immunohistochemical staining and western blotting. The growth inhibition effects in group 3 were compared with those in group 1 and 2, and the bystander effects and cell recoveries, after finishing the 5-FC treatment, evaluated until day 20 of the culture. RESULTS: The calculated survival percentages by from the MTT tests in the WiDr and LoVo cells revealed 110~150% enhancing effects after the administration of 5-FC concentration of 10, 100 and 1000micro M (group 1), and 30~90% after the administration of 5-FU at the same concentrations (group 2), which were shown to be statistically significantly (P<0.001). After transfection of pCMV/CD-1 plasmid vector into the WiDr and LoVo cells using the lipofectin method, the transfection rate of the WiDr and LoVo cells were 4.2 +/- 0.6% and 13.8 +/- 0.8%, respectively. Also the CD protein, with the polyclonal anti-CD antibody, by western blot, revealed weak expression on day 2, followed by strong expression on day 4, which progressively decreased by days 6 and 8 in the WiDr cells. Conversely, the LoVo cells showed weak expression on day 2, which progressively increased by days 4 and 6, was followed by the strongest expression on day 8 in LoVo cells. About 60~85% significant growth inhibition effects (P<0.001) were revealed in group 3, with proportionally different effects, corresponding to the CD gene transfection rate, concentration of 5-FC and duration of the culture. The 60~85% growth inhibition effects were complex of the response to the transfection rate and the bystander effects of CD expressed cells to the CD non-expressed cells. CONCLUSION: From our results, the administration of 5-FC after CD gene transfection revealed statistically significant growth inhibition effects of 60~85%, which were complex of the effects of CD expressed cells and the bystander effects to the CD non-expressed cells, and proportionally corresponding to the transfection rate, the duration of the CD protein expression and the concentration of 5-FC.

Transduction of yeast cytosine deaminase mediated by HIV-1 Tat basic domain into tumor cells induces chemosensitivity to 5-fluorocytosine

Enzyme/prodrug approach is one of the actively developing areas for cancer therapy. In an effort to develop more effective enzyme/prodrug systems, cell-permeable cytosine deaminase was produced by fusing yeast cytosine deaminase (yCD) in frame with RKKRRQRRR domain of HIV-1 Tat which is an efficient delivery peptide of the foreign proteins into cells. The purified Tat-yCD fusion protein expressed in Escherichia coli was readily transduced into mammalian cells in a time- and dose-dependent manner. A significant level of the transduced Tat-yCD protein was recovered in the cell and was stable for 24 h as indicated by both results of the enzymatic assay of 5-fluorocytosine (5-FC) conversion to 5-fluorouracil (5-FU) and Western blot analysis. The cells transduced with Tat-yCD become highly sensitive to the cytotoxicity of 5-FC, while cells treated with yCD are unaffected by 5-FC. In addition, a strong bystander effect was observed with conditioned media from cells transduced with Tat-yCD added to non-transduced cells. Tat-yCD fusion protein demonstrated here for its ability to transduce into cells and convert nontoxic prodrug 5-FC to the toxic antimetabolite 5-FU, may be a useful approach for cancer therapy.

Suicide Gene Therapy for Bladder Cancer Using a Recombinant Adenovirus Expressing Escherichia Coli Cytosine Deaminase / 대한비뇨기과학회지

PURPOSE: The poor prognosis of advanced bladder cancer requires the investigation of novel treatment modalities. In this study, we investigated the suicide gene therapy for bladder cancer, using the adenovirus-mediated expression of Escherichia coli cytosine deaminase (CD) in conjunction with the prodrug 5-fluorocytosine (5-FC). MATERIALS AND METHODS: A replication-deficient recombinant adenovirus, which contains the Rous sarcoma virus (RSV) promoter driving the expression of CD, (Ad-RSV-CD) was constructed. In vitro cell-killing assay, using Ad-RSV-CD (20 MOI) plus 5-FC (500muM), was performed in bladder cancer cell lines, HT-1376, UM-UC-3 and NBT-II. The CD enzymatic activity was measured in the Ad-RSV-CD (20 MOI) infected cells, and the concentrations of 5-fluorouracil (5-FU) yielding an IC50 were calculated for those cells. RESULTS: 5-FU dose response curve showed that IC50 of NBT-II was 0.8muM, HT-1376 1.0muM and UM-UC-3 5.1muM at day 6. The CD enzymatic activities of the Ad-RSV-CD infected UM-UC-3, HT-1376 and NBT-II cells were 5696, 4655, 1766 pmole/1x10(6) cells, respectively. Whereas the administration of 5-FC (500muM) or Ad-RSV-CD (20 MOI) alone demonstrated no cytotoxicity to cells, Ad-RSV-CD/5-FC exhibited a significant cytotoxic effect in the cells, especially the UM-UC-3 and HT-1376. CONCLUSIONS: Ad-RSV-CD/5-FC suicide gene therapy is effective for bladder cancer cells in cell cultures, suggesting this approach may have potential as a strategy for the treatment of bladder cancer.

The inhibitive effect of adenovirus mediated CD gene and 5-FC on proliferative human retinal pigment epithelialcells / 中华眼底病杂志

Objective To study the inhibitive effect of adenovirus mediated CD gene and 5-FC on proliferative human retinal pigment epithelial (HRPE) cells, and to search for an effective method to take precautions against proliferative vitroretinopathy (PVR). Method Different concentrations of CD and 5-FC were added respectively to the cultured third-growth-generation HRPE cells.Transferance rate was detected by positive HRPE cells marked by X-gal and LacZ. The number of HRPE cells were counted and evaluated by methylthiazol-tetrazollium (MTT) method. Results The adenovirus mediated CD gene could be transfered into HRPE cells with a dose-dependent manner. Positive HRPE cells with CD gene could transform 5-FC to 5-Fu,which could inhibit the increase of HRPE cells effectively. No obvious bystander effect on the growth of HRPE cells was detected. Conclusions The adenovirus may introduce a foreign gene into cultured HRPE cells efficiently. It could be a good method to treat and prevent PVR by medication.

EXPERIMENTAL STUDIES OF USING ADENOVIRUS-MEDIATED CD/5-FC SUICIDE GENE IN TREATMENT OF PAN- CREATIC CANCER / 解放军医学杂志

The purpose of this study was to evaluate the killing effects of CD/5-FC suicide gene system mediated by adenovirus vector on human pancreatic carcinoma. Recombinant adenoviruses containing cytosine deaminase(CD) gene were constructed by the homologous recombination method in bacteria. The newly recombinated Ad-CD containing green fluorescent protein (GFP) was propagated in 293 cells and purified by cesium chloride gradient centrifugatioa Human pancreatic carcinoma cell line was infected with this virus, then 5-FC was added, and XTT assay was used to estimate relative numbers of viable cells. A heterotopic human pancreatic adenocarcinoma was successfully established in the flank of Balb/c nude mice. Adenoviral liquid containing CD gene was directly injected into xenografts following administration of 5-FC to observe the antitumor effects. Positive clones were selected by using endonuclease to digest the recombinants and the concentration of viral liquid containing CD gene was 2 1011 pfu/ml. It was found that significant cytotoxic activities were possessed by 5-FC for CD gene-transduced pancreatic carcinoma cells, but there was little effect on nontransduced ones. The anticancer effect was seen in vivo in xenografts of nude mice with in situ CD gene transduction. Our conclusian is that CD gene mediated by adenovirus has a high infectivity and is efficient for gene therapy of pancreatic carcinoma. These data demonstrate the therapeutic efficacy of an enzyme prodrug strategy in treatment of experimental pancreatic

The Death Mechanism of LoVo Cells Transfected with Cytosine Deaminase Suicide Gene / 中国医师杂志

Objective To study the death mechanism of LoVo cells transfected with retroviral vector carrying cytosine deaminase (CD) suicide gene(G1CEACDNa). Methods Plasmid G1CEACDNa was transferred into the LoVo cells using liposomes method. RT-PCR was performed to detect CD mRNA expression using the total RNA extracted with TRIzol reagent. After exposed to 5-FC (1mmol?L -1 ), the cell growth inhibition rate and the “bystander effect” were determined by MTT, the cell ultramicroscopic structure was observed by electron microscope, and cell apoptosis was detected by flow cytometry. Results RT-PCR detection showed that there was 1.5kb band of CD product. The growth of the trasfected LoVo cells started to be inhibited after exposed 5-FC for 48h, and the inhibition rates at 72h,96h and 120h were 30%,50% and 80%, respetively.Electron microscope examination showed that there were apoptotic body. Meanwhile,a few cells showed necrosis alteration.Flow cytometry analysis showed that a few cells appeared apoptosis after exposed to 5-FC for 48h, the apoptotic rate and the necrosis rate at 72h and 96h were 23.8%, 35.1% and 20.4%, 26.0%, respectively. Conclusion The death mechanism of LoVo cells transfected with plasmid G1CEACDNa followed 5-FC treatment was involved in both necrosis and apoptosis. Apoptosis possibly was a mechanism of the bystander effect.

The therapeutic mechanism of adenovirus mediated CD/5-FC system in the treatment of oral squamous carcinoma cells / 实用口腔医学杂志

Objective: To study the therapeutic mechanism of adenovirus mediated cytosine deaminase /5-fluorocytosine (AdCMVCD/ 5-FC) suicide gene system in the treatment of oral squamous carcinoma cells in vitro. Methods: 3H-thymidine ( 3H-TdR) incorporation assay, flow cytometry (FCM), transmission electron microscope and TUNEL (TdT-mediated dUTP Nick End Labeling)assay were used to detected the changes of Tca8113 cells after the treatment with AdCMVCD/5-FC system. Results: After treatment with CD/5-FC system, 3H-TdR incorporation rate of the cells decreased and significantly decreased between different MOI (multiple of infection) at 5-FC 10 -3 mol/L (P0.05); many apoptotic cells were found under transmission electron microscope and the positive rate of apoptotic cells increased from (4.40?0.87)% (before treatment) to (15.80?1.55)% (P

The target effect of 5-flourocytosine combined thermotherapy on liver metastasis of colon cancer in nude mice / 中国普通外科杂志

Objective To investigate the target effect of cytosine deaminase genetherapy combined thermochemotherapy on liver metastasis of colon cancer in nude mice.Methods Plasmid G1CEACDNa were transferred into the human colon cancer LoVo cells by liposomes method.The models of liver metastasis of colon cancer were established by injected LoVo-CEACD into peritonum in 45 BALB/C nude mice,then which were randomly divided into three groups:The control(C)group;the prodrug therapy(PDT)group and the prodrug thermochemotherapy(PDTCT group)and 42 ℃ sodium chloride,38 ℃ 5-FC and 42 ℃ 5-FC were injected into the abdominal cavity [500 mg/(kg?d),for 21 d],respectively.After natural death of all mice,their life span,rate of liver metastasis and number of liver metastasis nodules were surveyed and compared among the three groups.The levels of the expression of target gene were detected by RT-PCR in carcinoma tissue,normal liver,stomach,lung,pancreas,small intestine and large intestine tissues.Pathological features were observed.Apoptotic index(AI)of tumor cells were analyzed in every group respectively.Results The CD gene was detected in the tumor tissues,but not in tissues of normal liver,stomach,lung,pancreas,small intestine and large intestine.In C,PDT and PDTCT group,the life span was(25.80?3.65),(34.27?4.08)and(41.87?3.91)d respectively;the rate of liver metastasis was 100.0%(15/15),40.0%(6/15)and 13.3%(2/15),respectively;the number of liver metastasis nodules was(2.93?1.16),(0.80?1.01)and(0.20?0.56),respectively;and AI of tumor cells was 4.59%,9.87%,17.4%,respectively.The life span of PDT and PDTCT guoup was significantly longer than that of group(P

Killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro / 中华消化杂志

Objective To evaluate the killing effects of cytosine deaminase (CD) gene mediated by adeno virus vector on human pancreatic cancer cell lines in vitro. Methods After CD gene was cloned into pAdTrack CMV CD, pAdTrack CMV CD and pAdEasy 1 were recombinated in bacteria. The newly recombinated Ad CD containing green fluorescent protein (GFP) was propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic cancer cell lines Patu 8988 and SW 1990 were infected with this virus, then 5 FC was added, XTT assay was used to estimate relative numbers of viable cell. Results The positive clones were selected by using endonuclease to digest the combinatants and the concentration of viral liquids containing CD gene was 2?10 11 PFU/ml. It was found that significant cytotoxic activities were possessed by 5 FC for CD gene transduced pancreatic cell lines, but little effects on the nontransduced pancreatic carcinoma cells. Conclusions CD gene mediated by adenovirus has a high infectivity and is efficient for gene therapy of pancreatic carcinoma cell lines. These data demonstrate the therapeutic efficacy of an enzyme as a prodrug strategy in experimental pancreatic cancer.

More Efficieat Induction of Autitumor Response by Combined Suicide Gene and Interleukin 2 Gene Transfer Mediated by Recombinant Aden-ovirus / 中国肿瘤生物治疗杂志

Antitumor effect of combined transfer of suicide gene and cytokine gene was evaluated in the present study. Adenoviruses expressing E. coli. cytosine deaminase (AdCD) and adenoviruses expressing murine interleukin 2 (AdTL2) were used for the treatment of tumor-bearing mice. The mice were inoculated s. c. with FBL-3 leukemia cells and 3 days later received intratumoral injection of AdCD in the presence or absence of AdIL2 followed by intraperitoneal 5-fluorocytosine (5FC) administration. The results demonstrated that tumor-bearing mice treated with AdCD/5FC in combination with AdTL2 showed more .potent inhibition of tumor growth and survived much longer as compared with mice treated with AdCD/5FC, AdEL2, AdlacZ/5FC or PBS. It was illustrated that the tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD4+ and CD8+ T cells infiltrated into the tumor after combined therapy. The splenic NK and CTL activities increased significantly in mice after combined transfer of CD gene and EH gene. Our results demonstrated that combined transfer of suicide gene and IL-2 gene could inhibit the growth of established tumor in mice significantly and induce antitumor immunity of the host efficiently.

Induction of Tumor Cell Apoptosis by Adenovirus-Mediated E. coli Cyto-sine Deaminase Gene Transfection and Its Bystander eftect / 中国肿瘤生物治疗杂志

Adenoviruses harboring E. coli cytosine deaminase gene (AdCD) were used to transfect murine FBL-3 ery-throleukemia cells in vitro. FBL3 cells infected with AdCD were more sensitive to 5-fluorocytosine (5-FC) than cells infected with a control adenovirus AdLacZ. Further study indicated that this combination therapy (AdCD and 5-FC) killed tumor cells by inducing apoptosis of FBL-3 cells. The supematants from FBL-3 cells treated with AdCD/5-Fc were transferred on the culture system of uninfected (wild - type) FBL-3 cells, the result indicated that only 6.25% of the supernatant could induce significant cytotoxicity on wild type FBL3 cells. The results demonoustrated that bystander effect plays an important role in AdCD-mediated cytotoxicities. Direct injection of AdCD into established subcutaneous FBL3 tumor in mice followed by daily intraperitoneal injection of 5-FC for 10 days was found to inhibit tumor growth significant-