OBJECTIVE: To construct nasopharyngeal carcinoma CNE-2 cell lines expressing stable fusion suicide gene CD/UPRT. UL49. METHOD: The plasmids of pcDNA3.1 (-)E6. BARF1p. CD/UPRT. UL49 was transfected into CNE-2 cells through lipofectamine, and the transfected CNE-2 cells were selected by G418 and prodrugs for getting the cells expressing fusion CD/UPRT. UL49 gene. The protein produced by the suicide gene was tested by Western-blotting in CNE-2 cells. RESULT: Suicide genes were expressed stably in CNE-2 cells. CONCLUSION We constructed nasopharyngeal carcinoma cell lines CNE-2 expressing stable suicide gene through lipofectamine.
Artificial Gene Fusion ; methods ; Carcinoma ; Cell Line, Tumor ; Genes, Transgenic, Suicide ; Humans ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics

Aeromonas hydrophila CGMCC 0911 possessing type I polyhydroxyalkanoate (PHA) synthase gene (phaC) only accumulate copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx), abbreviated as PHBHHx, from lauric acid as sole carbon source but not from glucose. The gene encoding type I PHA synthase was interrupted by insertion of a chloramphenicol resistance gene (Cm). Conjugation of suicide plasmid pFH10 transformed A. hydrophila CGMCC 0911 into a recombinant organism with the disrupted type I PHA synthase gene (phaC:: Cm) , through an in vivo homologous recombination process, type I phaC of A. hydrophila genome was replaced by the disrupted phaC, and Cm gene was integrated into the genome of A. hydrophila, resulting in type I phaC-negative mutant, which was proved by DNA sequencing. Results of GC analysis showed that this mutant could not accumulate PHBHHx again but accumulate medium-chain-length (mcl) PHA from lauric acid or glucose as carbon source, clearly indicating the existence of another type I PHA synthase in the wild type A. hydrophila. It will play its function and accumulate mcl PHA only when type I PHA synthase was inactivated. into the genome of A. hydrophila, resulting in type I phaC-negative mutant, which was proved by DNA sequencing. Results of GC analysis showed that this mutant could not accumulate PHBHHx again but accumulate medium-chain-length (mcl) PHA from lauric acid or glucose as carbon source, clearly indicating the existence of another type II PHA synthase in the wild type A. hydrophila. It will play its function and accumulate mcl PHA only when type I PHA synthase was inactivated.
Acyltransferases ; genetics ; metabolism ; Aeromonas ; enzymology ; genetics ; Bacterial Proteins ; genetics ; metabolism ; Genes, Transgenic, Suicide ; genetics ; Mutation ; Polyhydroxyalkanoates ; biosynthesis ; genetics

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

Gene therapy is one of the most attractive fields in tumor therapy. In past decades, significant progress has been achieved. Various approaches, such as viral and non-viral vectors and physical methods, have been developed to make gene delivery safer and more efficient. Several therapeutic strategies have evolved, including gene-based (tumor suppressor genes, suicide genes, antiangiogenic genes, cytokine and oxidative stress-based genes) and RNA-based (antisense oligonucleotides and RNA interference) approaches. In addition, immune response-based strategies (dendritic cell- and T cell-based therapy) are also under investigation in tumor gene therapy. This review highlights the progress and recent developments in gene delivery systems, therapeutic strategies, and possible clinical directions for gene therapy.
Dendritic Cells ; immunology ; Gene Transfer Techniques ; Genes, Transgenic, Suicide ; Genes, Tumor Suppressor ; Genetic Therapy ; methods ; Genetic Vectors ; Humans ; Neoplasms ; genetics ; therapy ; RNA Interference

Molecular nuclear imaging techniques are currently being developed to map the topography and level of gene expression following gene therapy. To date, two radionuclide-based imaging strategies have been investigated--using reporter genes encoding either intracellular enzymes or cell-surface receptors. In this article, we discuss these two reporter gene imaging systems that have been developed to detect gene expression noninvasively.
Gene Expression Regulation ; Gene Transfer Techniques ; Genes, Reporter ; genetics ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; Magnetic Resonance Imaging ; methods ; Molecular Biology ; Radionuclide Imaging ; Transfection

OBJECTIVE: To study the killing effect of suicide gene CDglyTK combined with GCV or 5-FC on the human laryngeal carcinoma Hep-2 cell line in vitro. METHOD: Constructed plasmid pcDNA3.1 (-) CMV. CDglyTK was verified by enzyme digestion of Xho I /Hind III and automatic sequence analysis, then it was introduced into Hep-2 cells by electroporation to yield cells expressing CDglyTK stably after selecting with G418(400 ng/L) for 14 da. The expression of CDglyTK mRNA in transfected Hep-2 cells was tested by RT-PCR. Compared with Hep-2 cells transferred with pcDNA3.1(-), in vitro chemosensitivity of CDglyTK-expressing Hep-2 cells to 5-FC, GCV or 5-FC + GCV was detected by MTT assay. RESULT: The recombinant plasmid contained full-length coding region sequence of CD and TK gene. A anticipated 707 bp fragment was amplified from total RNA of CDglyTK-expressing Hep-2 cells by RT-PCR and a fusion protein of 59 000 was detected in cell extract from transfected Hep-2 cells. In vitro study growth of CDglyTK-positive Hep-2 cells were inhibited by 5-FC, GCV or 5-FC + GCV respectively, and the antitumour effect of 5-FC + GCV is superior to 5-FC or GCV. CONCLUSION CDglyTK may be a candidate for treating human laryngeal cancer.
Cell Line, Tumor ; Cytosine Deaminase ; genetics ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Laryngeal Neoplasms ; genetics ; Plasmids ; Recombinant Fusion Proteins ; genetics ; Thymidine Kinase ; genetics

OBJECTIVETo investigate the selectively killing effect of adenovirus (Ad) mediated double suicide gene under the regulation of KDR promoter on vascular endothelial cells and colorectal tumor cells.

METHODS293 packaging cells were transfected with the plasmids of pAdEasy- KDR- CDglyTK and pAdEasy- CMV- CDglyTK and the infectious viruses were generated. The KDR expressive cells of ECV304,SW620 and the KDR inexpressive cells of LS174T were infected by two Ads. The infection rate was observed and the expression of CDglyTK was detected by RT- PCR. After treatment with different concentrations of 5- FC and GCV,the killing effect and bystander effect on ECV304,SW620 and LS174T were examined.

RESULTSThe titers of these two purified Ads were 2.0 x 10(12 ) pfu/ml. There was no significant difference in infection rate between two recombinant Ads infecting various cells,and the infection rate increased in accordance with the enhancing titers of Ads. RT- PCR demonstrated that there existed the product of CDglyTK gene in all the cells infected by Ad- CMV- CDglyTK and the cells infected by Ad- KDR- CDglyTK except in the SL174T. The curative effect in this system on various cells was shown as follows: (1) All cells infected with Ad- CMV- CDglyTK and some cells of ECV304 and SW620 infected with Ad- KDR- CDglyTK were highly sensitive to the prodrugs,but there was no significant differences among them (P > 0.05); compared with ECV304 and SW620 cells,LS174T cells were not sensitive to the two prodrugs (P< 0.001). (2) The efficacy of double suicide gene was better than that of single suicide gene (P< 0.001). (3) The system had considerable bystander effect.

CONCLUSIONThe double suicide gene under the regulation of KDR promoter has specific killing effect on the KDR- expressing endothelial cells and colorectal tumor cells.

Adenoviridae ; genetics ; Cell Line, Tumor ; Endothelial Cells ; cytology ; Gene Expression Regulation ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Humans ; Promoter Regions, Genetic ; Vascular Endothelial Growth Factor Receptor-2 ; genetics

OBJECTIVETo investigate the effect of herpes simplex virus-thymidine kinase (HSV-TK) gene transduced into T cell clone on the treatment of ulcerative colitis (UC) in rat.

METHODSThe T cell clone transduced with HSV-tk was infused into 27 rats with UC. Changes of stimulation index (SI), CD4(+), CD8(+), IL-13, IL-4 were detected, and pathological changes before and after infusion was compared.

RESULTSIn the second and third day after tk+ T clone infusion, the inflammation of colon was assimilated. Two weeks later, the colon began to renovate and mend. The average SI was 7.39+/-1.24 before infusion, and 2.67+/-0.87 after infusion (P<0.05). Peripheral blood levels of CD4(+), CD8(+) or IL-13 and IL-4 in therapeutic group were significantly decreased as compared to control group (P<0.05).

CONCLUSIONHSV-tk gene transduced into T lymphocyte clone and infused back is effective for UC gene therapy and target design in rat.

Animals ; Colitis, Ulcerative ; therapy ; Genes, Transgenic, Suicide ; Genetic Therapy ; Male ; Rats ; Rats, Wistar ; Simplexvirus ; genetics ; T-Lymphocytes ; Thymidine Kinase ; genetics ; Viral Proteins ; genetics

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

In order to investigate the application of recombinant adeno-associated virus (rAAV) vector containing Tet regulation system and HSVtk gene in cancer gene therapy, pAAV/TRE/HSVtk/Tet-On was constructed and identified with PCR and restriction enzyme digestion. Packaging cells HEK293 were cotransfected with plasmids pAAV/TRE/HSVtk/Tet-On, pAAV-RC and pAAV-helper to produce infectious rAAV, and CsCl2 densitygradient centrifugation method was performed for purification and concentration of rAAV. The viruses were then transduced into MCF-7 cells. The results of dot blot hybridization indicate that the rAAV can transfer the target gene into MCF-7 cells. MTT assay showed that GCV could kill AAV-infected MCF-7 cells under the induction of Dox. The data demonstrated that rAAV containing Tet regulation system and HSVtk gene was successfully obtained, and could be used for further investigation of in vivo and in vitro experiments.
Cell Line, Tumor ; Dependovirus ; genetics ; metabolism ; Doxycycline ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; genetics ; Transfection