BACKGROUNDCytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic beta-alanine without uracil phosphoribosyltransferase (UPRT). UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD::UPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD::UPRT/5-FC has not been verified in vivo and in vitro. Before the CD::UPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using (19)F-magnetic resonance spectroscopy ((19)F-MRS) and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect.

METHODSC6 and C6-CD::UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by (19)F-MRS. Rats with intracranial xenografted encephalic C6-CD::UPRT glioma were injected intraperitoneally with 5-FC and their (19)F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD::UPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CD::UPRT cells were examined by (19)F-MRS. A comparative study of mean was performed using analysis of variance (ANOVA).

RESULTS(19)F-MRS on samples from C6-CD::UPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides (F-Nuctd). For the C6 mixture, only the 5-FC peak was detected. In vivo serial (19)F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about 1 hour, reached a maximum at around 160 minutes, and was detectable for several hours. At a 10% ratio of C6-CD::UPRT cells, the survival rate was (79.55 +/- 0.88)% (P < 0.01). As the C6-CD::UPRT ratio increased, the survival rate of the cells decreased. (19)F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD::UPRT in the mixture increased.

CONCLUSIONS(19)F-MRS studies indicated that C6-CD::UPRT cells could effectively express CD and UPRT enzymes. The CD::UPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CD::UPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and (19)F-MRS can monitor transferred CD::UPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.

Animals ; Antimetabolites ; pharmacokinetics ; therapeutic use ; Cell Line ; Cytosine Deaminase ; genetics ; physiology ; Flucytosine ; pharmacokinetics ; therapeutic use ; Genetic Therapy ; methods ; Glioma ; drug therapy ; therapy ; Humans ; Magnetic Resonance Imaging ; Male ; Pentosyltransferases ; genetics ; physiology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the anticancer activity of three-pronged approach: liposome transfer cytosine deaminase (CD) gene, ionizing radiation, and dendritic cells (DCs).

METHODSAfter rectal cancer cells were killed by CD and 5-FC and radiotherapy with improved liposome transfection and radiosensitization, the killed cancer cells, DCs and T lymphocytes were incubated together. The uptake of (3)H-TdR weighed the immune activity of DCs. The effect of anticancer was monitored by MTT.

RESULTSDCs acquired the apoptosis body and other antigen from the killed cancer cells and presented them to T-lymphocytes. The induced CTL killed the cancer cells powerfully and selectively. The mortality of rectal cancer cells was 98.1%, 76.2%, 37.8% at the ratio of efficacy to target of 30:1, 15:1, 1:1, respectively.

CONCLUSIONThe three-pronged approach may represent a powerful and safe means to selectively destroy cancer cells.

Combined Modality Therapy ; Cytosine Deaminase ; Dendritic Cells ; Gamma Rays ; therapeutic use ; Genetic Therapy ; Humans ; In Vitro Techniques ; Liposomes ; Nucleoside Deaminases ; genetics ; therapeutic use ; Radiation, Ionizing ; Rectal Neoplasms ; mortality ; radiotherapy ; therapy

OBJECTIVESTo evaluate the efficacy of antitumour colon cancer cell vaccine modified by escherichia coli cytosine deaminase (EC-CD).

METHODSMouse colon cancer cell vaccine CT26/CD was established by gene modification using retrovirus plasmid pLCDSN. Its tumorigenicity and effect on liver metastasis model established with wild-type colon cancer were evaluated.

RESULTSCT26/CD was established successfully and proliferated in medium containing 0.6 g/L G418 stably. EC-CD gene expression on these mutant cells was confirmed by RT-PCR. These mutant cells were more sensitive to 5-fluorocytosine (5-FC) compared with the wild-type ones (P=0.000), and presented excellent bystander effect. CT26/CD had the same tumorigenicity as its parental cells (P=0.892). CT26/CD, combined with the prodrug 5-FC, could inhibit tumor progress and live metastasis, and prolonged the survival of the liver metastasis model animals (P=0.000).

CONCLUSIONThe colon cancer cell vaccine modified by EC-CD presented anti-tumor effect in vivo, when treated with the prodrug.

Amino Acid Motifs ; Animals ; Cancer Vaccines ; therapeutic use ; Cell Line, Tumor ; Colonic Neoplasms ; therapy ; Cytosine Deaminase ; genetics ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; genetics ; Humans ; Mice ; Mice, Inbred BALB C

OBJECTIVETo study the antitumor and distant bystander effects of cationic liposome-mediated cytosine deaminase (CD)/5-fluorocytosine (5-FC) suicide gene system combined with interferon-gamma (IFN-gamma) in vivo.

METHODSMurine hepatoma 22 (H22) cells transfected by CD gene were inoculated subcutaneous in Kunming mice in the left axillary region, and the H22 cells without CD gene transfection were inoculated in the right axillary region. The mice were randomly divided into 4 groups and treated with normal saline , 5-FC, IFN-gamma, and 5-FC+ IFN-gamma on a daily basis. The tumor inhibition and distant bystander effects were observed in the mice.

RESULTSExposure of CD gene-transfected tumor to 5-Fc resulted in obvious tumor growth inhibition with an inhibition rate of 78.38%, which was significantly increased to 93.21% (P<0.01) with 5-Fc +IFN-gamma treatment. A notable distant bystander effect in the CD/5-FC suicide gene system was observed in vivo, with a tumor inhibition rate of was 54.42%; when combined with IFN-gamma, the inhibition rate increased significantly to 87.57% (P<0.05).

CONCLUSIONWhen combined with IFN-gamma, CD/5-FC suicide system has stronger anti-tumor and distant bystander effects. CD/5-FC suicide gene system combined with IFN-gamma may provide a potential therapy for malignant tumors.

Animals ; Bystander Effect ; Cations ; chemistry ; Cytosine Deaminase ; genetics ; Flucytosine ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Interferon-gamma ; therapeutic use ; Liposomes ; Liver Neoplasms, Experimental ; therapy ; Male ; Mice ; Random Allocation

OBJECTIVETo investigate the therapeutic effect and metabolism of 5-fluorocytosine (5-FC) in human tongue squamous carcinoma cells after treatment with adenovirus-mediated cytosine deaminase (AdCMVCD)/5-FC system.

METHODSHuman tongue squamous carcinoma cells (Tca8113 cell line) and its xenografts in BALB/c nude mice were treated with AdCMVCD/5-FC system. The killing effect in vitro and bystander effect were detected by microculture tetrazolium (MTT) assay. Tumor inhibition effect and histopathological changes were observed in vivo. High-performance liquid chromatography (HPLC) was performed to determine the metabolism of 5-FC in vitro and in vivo.

RESULTSAdCMVCD/5-FC system had strong killing effect and bystander effect on Tca8113 cells. Both condition media and cell extracts showed two peaks identified as 5-FC and 5-fluorouracil (5-FU) by HPLC and a time-dependent generation of 5-FU and concomitant time-dependent decreases of 5-FC. Compared to the control groups, mice treated with AdCMVCD/5-FC system demonstrated significant tumor regression (P < 0.001); the tumor doubling time prolonged and inhibition rate was 92.62%. There were substantial tumor necrotic areas and infiltrative lymphocytes around necrotic areas in the AdCMVCD/5-FC treated group under light microscope. There was a significantly low concentration of 5-FC and high concentration of 5-FU in tumor tissue, but only 5-FC was found in blood.

CONCLUSIONAdCMVCD/5-FC suicide gene system had significant in vitro and in vivo anti-tumor effect on human tongue squamous cell carcinoma due to convert 5-FC into 5-FU.

Adenoviridae ; genetics ; Animals ; Carcinoma, Squamous Cell ; pathology ; therapy ; Cytosine Deaminase ; Female ; Flucytosine ; metabolism ; therapeutic use ; Genetic Therapy ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Nucleoside Deaminases ; genetics ; Tongue Neoplasms ; pathology ; therapy ; Transplantation, Heterologous ; Tumor Cells, Cultured

OBJECTIVETo evaluate the feasibility and efficacy of cytocine deaminase-thymidine kinase (CD-TK) fusion double suicide gene therapy using adenovirus mediated CD-TK gene and green fluorescent rotein (GFP) gene combined with ganciclovir(GCV) or 5-flourocytosine(5-FC) in a murine subcutaneous bladder carcinoma model.

METHODSA replication defective adenovirus vector containing CD-TK gene was used. Subcutaneous tumors were established in syngenic C57BL/6 female mice with 1 x 10(6) Mb49 cells. Intratumoral injection of AdCD-TK (1.58 x 10(8) PFU, qd x days) in combination with GCV (40 mg.kg(-1).d(-1), ip, qd x 10 days) or 5-FC (400 mg.kg(-1).d(-1), ip, qd x 10 days) was administered in vivo for the determination of treatment efficacy in separate controlled experiments.

RESULTSIn vivo experiments demonstrated that the mean volume of tumor in the group of AdCD-TK/GCV(326.58+/-109.56 mm(3)), AdCD-TK/5-FC (235.33+/-62.94 mm(3)) and AdCD-TK/(GCV+5-FC) (23.58+/-6.78 mm(3)) was reduced significantly compared with that of control group (993.51+/-158.32 mm(3)) (P=0.00), the mean volume of tumor in the group of AdCD-TK/(GCV+5-FC) was significantly less than that in the group of AdCD-TK/GCV or AdCD-TK/5-FC (P=0.04). Tumor necrosis was revealed by histomorphology compared with control animals.

CONCLUSIONSAdenovirus mediated CD-TK double suicide gene combining with GCV or 5-FC could provide an effective therapy in an experimental murine bladder carcinoma by significantly inhibiting tumor growth. The treatment efficacy of AdCD-TK combining GCV and 5-FC was superior to that of AdCD-TK combining GCV or AdCD-TK combining 5-FC.

Adenoviridae ; genetics ; Animals ; Cell Line ; Cell Line, Tumor ; Cytosine Deaminase ; genetics ; metabolism ; Defective Viruses ; genetics ; Female ; Flucytosine ; pharmacology ; therapeutic use ; Ganciclovir ; pharmacology ; therapeutic use ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Thymidine Kinase ; genetics ; metabolism ; Treatment Outcome ; Urinary Bladder Neoplasms ; pathology ; therapy

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.
Animals ; Antimetabolites/*metabolism/pharmacology ; Bystander Effect ; Cytosine Deaminase/genetics/*metabolism ; Flucytosine/*metabolism/pharmacology ; Gene Products, tat/chemistry/genetics/*metabolism ; Genetic Vectors/genetics/metabolism ; HIV-1/metabolism ; Hela Cells/drug effects/physiology ; Humans ; Prodrugs/metabolism/therapeutic use ; Recombinant Fusion Proteins/genetics/*metabolism ; Research Support, Non-U.S. Gov't ; Saccharomyces cerevisiae Proteins/genetics/*metabolism ; *Transduction, Genetic

OBJECTIVETo investigate the effect of cytosine deaminase (CD) gene plus 5-fluorocytosine (5-Fc) on the growth of human colon cancer xenograftin nude mice.

METHODSRetroviral vector expressing CD gene was transfected into human colon cancer SW1116 cells. Expression of the transfected CD gene in SW1116 (SWCD(2)) was confirmed by RT-PCR. The cytotoxicity of 5-Fc on SW1116 was determined by MTT assay in vitro. In vivo, the CD gene expression vector was injected intratumorally and 5-Fc was given by ip injections.

RESULTSIn vitro, SWCD(2) cells were killed by 5-Fc with an IC(50) of 66 micromol/L while the nontrasfected SW1116 cells needed an IC(50) of 16 000 micromol/L to be killed. The growth of SWCD(2) xenografts was significantly inhibited by systemic administration of 5-Fc.

CONCLUSIONCD gene/5-Fc system is a potential gene therapy strategy for human colon cancer.

Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; metabolism ; pathology ; prevention & control ; Combined Modality Therapy ; Cytosine Deaminase ; genetics ; metabolism ; Female ; Flucytosine ; pharmacology ; therapeutic use ; Genetic Therapy ; Genetic Vectors ; Humans ; Inhibitory Concentration 50 ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Retroviridae ; genetics ; Transfection

Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation.
Adolescent ; Animals ; Cell Death/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Transformation, Neoplastic/drug effects/pathology ; Child ; Cytosine Deaminase/*genetics/therapeutic use ; Fluorouracil/pharmacology ; Genetic Therapy ; Genomic Instability/drug effects ; Humans ; Karyotype ; Mesenchymal Stromal Cells/*cytology/drug effects/metabolism ; Mice ; Multipotent Stem Cells/cytology/drug effects/metabolism ; Neoplasms/therapy ; Retroviridae/*metabolism ; Time Factors ; *Transduction, Genetic