No abstract available.
Cytosine Deaminase* ; Cytosine* ; Genetic Therapy* ; Osteocalcin* ; Prostate* ; Prostatic Neoplasms*

No abstract available.
Bone Marrow* ; Cytosine Deaminase* ; Cytosine* ; Homicide* ; Mesenchymal Stromal Cells* ; Prostate* ; Prostatic Neoplasms*

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.
Cytosine Deaminase ; Genes, vif ; Genetic Therapy* ; Herpesvirus 1, Human ; Nuclear Medicine ; Oncogenes ; Thymidine Kinase

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.
beta-Galactosidase ; Blotting, Western ; Breast ; Bystander Effect ; Cell Line* ; Colon* ; Colonic Neoplasms* ; Colorectal Neoplasms ; Cytosine Deaminase* ; Cytosine* ; Eukaryotic Cells ; Flucytosine* ; Fluorouracil ; Genes, Neoplasm ; Glioblastoma ; Homicide* ; Plasmids ; Prostate ; Transfection*

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.
beta-Galactosidase ; Blotting, Western ; Breast ; Bystander Effect ; Cell Line* ; Colon* ; Colonic Neoplasms* ; Colorectal Neoplasms ; Cytosine Deaminase* ; Cytosine* ; Eukaryotic Cells ; Flucytosine* ; Fluorouracil ; Genes, Neoplasm ; Glioblastoma ; Homicide* ; Plasmids ; Prostate ; Transfection*

OBJECTIVETo investigate whether hyperthermia can enhance the killing effect of 5- fluorocytosine (5- FC) on human colorectal carcinoma cell lines SW480 transfected with carcinoembryonic antigen (CEA) tissue- specific cytosine deaminase (CD) gene in vitro,and study its mechanism.

METHODSHuman colorectal carcinoma cell lines SW480 transfected with G1CEACDNa were cultured. The proliferated colonies were treated with the combined therapy of 5-FC and hyperthermia at a temperature of 43 degrees C for 30 min. After eight days, MTT was used to calculate the cellular survival rate,to analyze the killing effect of 5-FC combined with hyperthermia on SW480 cells transfected with CD gene. Flow cytometry was performed to analyze the cellular cycle and transmission electron microscope was used to observe the morphologic changes of SW480 cells after thermochemotherapy.

RESULTSHyperthermia combined with 5-FC had an enhanced killing effect on SW480-CEACD cells than 5-FC alone (P< 0.05, t =2.403, n=9). Flow cytometry revealed that the proportion of S stage cell increased in the group treated with hyperthermia and 5- FC (P< 0.001, t =7.158, n=6). Transmission electron microscope showed apoptosis after thermo- chemotherapy.

CONCLUSIONSHyperthermia can improve the anti- tumor effect of 5- FC on human colorectal carcinoma cell lines SW480 transfected with CD gene, and the cells were blocked at S stage of cellular cycle and apoptosis was induced following thermochemotherapy.

Cell Line, Tumor ; Cytosine Deaminase ; genetics ; Flucytosine ; pharmacology ; Genes, Transgenic, Suicide ; Genetic Therapy ; methods ; Hot Temperature ; Humans

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.
Adenoviridae* ; Cell Culture Techniques ; Cell Line ; Cytosine Deaminase* ; Cytosine* ; Escherichia coli* ; Escherichia* ; Flucytosine ; Fluorouracil ; Genetic Therapy* ; Inhibitory Concentration 50 ; Prognosis ; Rous sarcoma virus ; Suicide* ; Urinary Bladder Neoplasms* ; Urinary Bladder*

PURPOSE: Genetically engineered stem cells may be advantageous for gene therapy against various human cancers due to their inherent tumor-tropic properties. In this study, genetically engineered human neural stem cells (HB1.F3) expressing Escherichia coli cytosine deaminase (CD) (HB1.F3.CD) and human interferon-β (IFN-β) (HB1.F3.CD.IFN-β) were employed against lymph node–derived metastatic colorectal adenocarcinoma. MATERIALS AND METHODS: CD can convert a prodrug, 5-fluorocytosine (5-FC), to active 5-fluorouracil, which inhibits tumor growth through the inhibition of DNA synthesis,while IFN-β also strongly inhibits tumor growth by inducing the apoptotic process. In reverse transcription polymerase chain reaction analysis, we confirmed that HB1.F3.CD cells expressed the CD gene and HB1.F3.CD.IFN-β cells expressed both CD and IFN-β genes. RESULTS: In results of a modified trans-well migration assay, HB1.F3.CD and HB1.F3.CD.IFN-β cells selectively migrated toward SW-620, human lymph node–derived metastatic colorectal adenocarcinoma cells. The viability of SW-620 cells was significantly reduced when co-cultured with HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. In addition, it was found that the tumor-tropic properties of these engineered human neural stem cells (hNSCs) were attributed to chemoattractant molecules including stromal cell-derived factor 1, c-Kit, urokinase receptor, urokinase-type plasminogen activator, and C-C chemokine receptor type 2 secreted by SW-620 cells. In a xenograft mouse model, treatment with hNSC resulted in significantly inhibited growth of the tumor mass without virulent effects on the animals. CONCLUSION: The current results indicate that engineered hNSCs and a prodrug treatment inhibited the growth of SW-620 cells. Therefore, hNSC therapy may be a clinically effective tool for the treatment of lymph node metastatic colorectal cancer.
Adenocarcinoma* ; Animals ; Chemokine CXCL12 ; Colorectal Neoplasms ; Cytosine Deaminase* ; Cytosine* ; DNA ; Escherichia coli ; Flucytosine ; Fluorouracil ; Genetic Therapy ; Heterografts* ; Humans ; Interferon-beta ; Lymph Nodes ; Lymphatic Metastasis ; Mice* ; Neural Stem Cells* ; Polymerase Chain Reaction ; Reverse Transcription ; Stem Cells ; Urokinase-Type Plasminogen Activator

OBJECTIVETo determine the efficacy of adenovirus mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer.

METHODSCytosine Deaminase(CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria. The newly recombined adenovirus (Ad)-CD containing green fluorescent protein (GFP) were packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line-Patu8988 was infected with this virus, then 5FC was added. XTT assay was used to estimate relative numbers of viable cells. In vivo model of pancreatic cancer was established by injecting 1.0 x 10(7) Patu8988 cells subcutaneously in Balb/c nude mice. When tumors were palpable, Ad-CD was injected into each tumor and 5FC was administered.

RESULTSPositive clones were selected using endonuclease to digest the recombinants and the concentration of viral liquids containing the CD gene was 2 x 10(11) pfu /ml. Significant cytotoxic activity as shown for 5FC in the CD gene transduced 8988 cell line, while little effect was found in the nontransduced pancreatic carcinoma cells. Antitumor effect was observed in Patu8988 xenograft nude mice with in situ CD gene transduction.

CONCLUSIONSCD gene mediated by adenovirus has high infectivity and may be useful for gene therapy in pancreatic carcinoma. These data demonstrate the use of an enzyme prodrug strategy in experimental pancreatic cancer.

Adenoviridae ; genetics ; Animals ; Cytosine Deaminase ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleoside Deaminases ; genetics ; Pancreatic Neoplasms ; therapy

OBJECTIVETo evaluate the toxic effects of the CD-TK fusion gene systems against prostate carcinoma cell line RM-1 for assessing the value of suicidal gene therapy for prostate carcinoma.

METHODSCD-TK fusion gene and green fluorescent protein (GFP) gene were transfected into RM-1 cells through adenovirus vectors. RT-PCR was used to demonstrate successful transfection and transcription of the suicidal genes. The toxic effects of 5-FC and GCV used alone or in combination on the transfected cells were observed by MTT assay, with the non-transfected RM-1 cells serving as control.

RESULTSCytotoxic activity of CD/5-FC and TK/GCV systems against RM-1 cells was observed, and combined treatment with the two drugs resulted in significantly lowered survival of CD-TK-expressing cells (P<0.05). After exposure to 5-FC and GCV for 72 h, the survival rate of the transfected cells decreased to 71.56% and 47.27%, respectively, and their combined use resulted in a survival rate as low as 18.46%.

CONCLUSIONCD-TK fusion double suicidal gene system can produce significantly stronger toxic effect against RM-1 cells in vitro than either of suicidal genes.

Cell Line, Tumor ; Cytosine Deaminase ; pharmacology ; Genes, Transgenic, Suicide ; Genetic Therapy ; methods ; Genetic Vectors ; Humans ; Male ; Prostatic Neoplasms ; therapy ; Reverse Transcriptase Polymerase Chain Reaction ; Thymidine Kinase ; pharmacology ; Transfection