Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.
Antiviral Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cells, Cultured ; Dependovirus ; genetics ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Ganciclovir ; pharmacology ; Gene Expression Regulation, Neoplastic ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; genetics ; Humans ; In Situ Nick-End Labeling ; Luciferases ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics ; Pulmonary Alveoli ; cytology ; metabolism ; Pulmonary Surfactant-Associated Protein A ; genetics ; metabolism ; Thymidine Kinase ; genetics ; metabolism

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

Constructing robust gene circuits is a fundamental work for synthetic biology. Bacteria with suicide gene circuit based on quorum-sensing will kill themselves in a controllable pattern upon certain cell density. In the media of different IPTG inducer concentration, we observed the growth and suicidal behavior of the Escherichia coli. Top10F' with such gene circuit, screened the mutants and determined their mutated loci. The results show that, with higher IPTG concentration, the more wild type bacteria were killed; as well the mutants emerged earlier and spread over the population more quickly. The sequence of plasmids in those mutants revealed that a transposon inserted into the luxR gene and therefore disrupted Quorum-Sensing of these individuals. Furthermore, the insertion sequence of the plasmid can solely result in the mutants escaping from suicide.
Culture Media ; chemistry ; DNA Transposable Elements ; genetics ; Escherichia coli ; genetics ; growth & development ; Gene Expression Regulation, Bacterial ; Genes, Synthetic ; genetics ; Genes, Transgenic, Suicide ; Isopropyl Thiogalactoside ; chemistry ; Mutation ; Quorum Sensing ; genetics ; Repressor Proteins ; genetics ; Trans-Activators ; genetics

OBJECTIVETo investigate the selective killing effect of herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene system controlled by human IGF-II P4 promoter on HCC cells in vitro.

METHODSRecombinant shuttle plasmid vectors driven by IGF-II P4 promoter and driven by CMV promoter were constructed by techniques of gene recombination. The recombinant shuttle plasmids were then transfected into HepG2 and HeLa cells by techniques of lipidosome transfection. EGFP expression was detected by fluoroscopy. Tk and EGFP mRNA expression were detected by RT-PCR. The selective killing effect after GCV application was determined with MTT method. Statistical analysis was performed with ANOVA analysis.

RESULTSIdentification of pDC316-tkEGFP-P4 by enzyme digestion and sequencing analysis showed that the construction of the recombinant shuttle plasmid was correct. It was found that green fluorescence protein could only be seen in HepG2 cells, not in HeLa cells. The results of RT-PCR showed only two bands could be seen in the samples of pDC316-tkEGFP-P4 transfected HepG2 cells. The growth of HepG2 cells transfected with pDC316-tkEGFP-CMV and pDC316-tkEGFP-P4 were inhibited remarkably, the growth inhibition rates were 6.95% +/- 0.67%, 24.99% +/- 1.53%, 49.68% +/- 1.68%, 71.85% +/- 3.28% and 4.83% +/- 0.35% vs 17.34% +/- 1.15%, 30.17% +/- 1.30%, 40.39% +/- 0.82% (F = 24.055, P < 0.05), respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-CMV were also inhibited, the growth inhibition rates were 6.36% +/- 0.83%, 23.95% +/- 1.72%, 45.13% +/- 1.64% and 69.38% +/- 3.17%, respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-P4 was not inhibited. The growth inhibition rates were 0.91 +/- 0.04, 1.18 +/- 1.32, 1.19 +/- 0.10 and 1.32 +/- 0.05 (F = 26.469, P < 0.01) , respectively.

CONCLUSIONThe shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-II P4 promoter has been constructed successfully and its specific expression in HepG2 cells provided the basis for targeted gene therapy for HCC.

Cell Line, Tumor ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; Humans ; Insulin-Like Growth Factor II ; genetics ; pharmacology ; Plasmids ; Promoter Regions, Genetic ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVETo explore the synergistic anti-tumor effect of radiotherapy and horseradish peroxidase/prodrug indole-3-acetic acid (HRP/IAA) gene therapy system using chimeric hTERT promoter responsive to ionizing radiation.

METHODSThe synthetic hTERT promoters containing four tandem-repeat copies of radio-inducible CArG elements, and the chimeric promoter containing cytomegalovirus (CMV) early promoter were both constructed. The activities of the chimeric promoters in cancer cell lines (HeLa, A549, and MHCC97) and normal cell line (MRC-5) were detected using luciferase reporter gene expression analysis after a (60)Co γ-irradiation treatment at a series of doses(a single dose of 0 to 10 Gy). The anti-tumor effect of combining irradiation with HRP/IAA gene-directed enzyme prodrug therapy system controlled by the chimeric promoter was tested by colony formation assay, cell counting and apoptosis analysis.

RESULTSThe chimeric promoters were ineffective in normal human cells, even after irradiation, but the expression of luciferase gene in tumor cells was significantly higher. The activity of the chimeric promoter in MRC-5 cells was 22.3%, 12.9% and 13.6% of that in HeLa, A549 and MHCC97 cells, respectively. After irradiation, the ratios were 11.7%, 8.7% and 8.8%, respectively. Furthermore, the chimeric promoters could successfully induce the expression of luciferase gene following different doses of radiation, with maximal inducible activity seen after 6 Gy irradiation. The chimeric promoter containing four tandem-repeat copies of radio-inducible CArG elements and CMV early promoter showed the highest activity with 6 Gy irradiation. The relative luciferase activities in HeLa, A549 and MHCC97 cells were 1.7 ± 0.2, 2.3 ± 0.2 and 2.3 ± 0.1, respectively. The chimeric promoter mediated suicide gene therapy system could increase radio-sensitivity in different cancer cells. Compared with the control system, it plus irradiation showed stronger cell proliferation inhibition, 67.3% vs. 26.1% in HeLa, 69.0% vs. 28.3% in A549, 64.6% vs. 20.8% in MHCC97 cells, and also higher apoptosis-inducing effect, 39.6% vs. 14.2% in HeLa, 33.0% vs. 12.4% in A549, and 33.2% vs. 14.2% in MHCC97 cells.

CONCLUSIONSChimeric promoter containing hTERT promoter, CArG elements and CMV promoter preserve the tumor-specificity in telomerase-positive tumor cells, and irradiation-responsive to low dose of radiation. The suicide gene therapy using this promoter plus radiotherapy show a strong anti-tumor effect in vitro. It is expected to have a good potential for future application in gene radiotherapy.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Combined Modality Therapy ; Cytomegalovirus ; genetics ; Genes, Transgenic, Suicide ; Genetic Therapy ; methods ; Genetic Vectors ; Horseradish Peroxidase ; genetics ; metabolism ; Humans ; Indoleacetic Acids ; metabolism ; Luciferases ; genetics ; metabolism ; Plasmids ; Prodrugs ; Promoter Regions, Genetic ; radiation effects ; Radiotherapy ; methods ; Recombinant Proteins ; genetics ; metabolism ; Telomerase ; genetics ; metabolism ; Transfection

OBJECTIVEThe aim of this study was to construct a recombinant plasmid carrying fusion suicide gene CDglyTK and RNA interference eukaryotic expressing vector targeting to STAT3, and to investigate the effect of double suicide gene combined with RNAi targeting to STAT3 on HCT116 and HUVEC cells in vitro.

METHODSThe CD and TK were cloned by polymerase chain reaction (PCR), and fusion gene CDglyTK was inserted into plasmid pEGFP after DNA sequence analysis, enzyme digestion and ligation. The recombinant plasmid was analyzed by PCR amplification and electrophoresis and enzyme digestion. DNA sequences containing small hairpin structure targeting to STAT3 were synthesized and inserted into the vector. The CDglyTK gene expressions in HCT116 and HUVEC cells were examined by reverse transcription-polymerase chain reaction (RT-PCR) after transfection of HCT116 and HUVEC cells. The inhibitory effect of RNA interference vector targeting to STAT3 was analyzed by RT-PCR and Western blot. The effects of 5-FC and GCV on HCT116 and HUVEC cells transfected with the recombinant plasmids were detected by MTT staining.

RESULTSThe results of restriction enzyme digestion and PCR amplification and electrophoresis showed that the recombinant pEGFP/CDglyTK was constructed correctly. The mRNA expression of gene CDglyTK was detected in HCT116 and HUVEC cells which transfected with the recombinant plasmid. The results of RT-PCR and Western blot showed that the RNA interference expression vector targeting to STAT3 effectively inhibited the expression of STAT3 in HCT116 cells. The results of MTT test showed that the inhibition ratio of group pEGFP/CDglyTK was (63.72 ± 0.64)%, significantly higher than that of control group (P < 0.05). The inhibition rate of group pEGFP/STAT3 siRNA was (47.02 ± 0.39)%, which was lower than that of group pEGFP/CDglyTK (P < 0.05), and higher than that of control group (P < 0.05). The inhibition rate of group pEGFP/CdglyTK + pEGFP/STAT3 siRNA was (85.10 ± 0.17)%, significantly higher than those of groups pEGFP/CDglyTK and group pEGFP/STAT3 siRNA (P < 0.05). Meanwhile, in HUVEC cells, the inhibition rate of group pEGFP/CDglyTK was (70.24 ± 0.33)%, significantly higher than that of the control group (P < 0.05). The inhibition rate of group pEGFP/STAT3 siRNA was (46.32 ± 0.15)%, significantly lower than that of group pEGFP/CdglyTK (P < 0.05), and higher than that of the control group (P < 0.05). The inhibition rate of group pEGFP/CdglyTK+pEGFP/STAT3 siRNA was (87.10 ± 0.24)%, significantly higher than those of groups pEGFP/CDglyTK and pEGFP/STAT3 siRNA(P < 0.05).

CONCLUSIONThe recombinant plasmids pEGFP-CDglyTK and pEGFP/STAT3 siRNA have inhibitory effect on HCT116 and HUVEC cells. The killing effects of double suicide gene combined with RNAi targeting to STAT3 are much better than those of single gene therapy.

Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; therapy ; Genes, Transgenic, Suicide ; Genetic Therapy ; Genetic Vectors ; Humans ; In Vitro Techniques ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; STAT3 Transcription Factor ; genetics ; Transfection

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

OBJECTIVE: To determine the effect and molecular mechanism of DNA damage caused by suicide gene therapy system HSV-TK/GCV under Tet-On regulation in human breast cancer cell line MCF-7 infected by recombinant adeno-associated virus (rAAV). METHODS: We used comet assay to detect the effect of HSV-TK/GCV suicide gene regulation system on MCF-7 DNA damage, and analyzed the expression change of relative DNA damage response active genes and proteins with RT-PCR and Western blot. RESULTS: Compared with other control groups, the comet assay showed that MCF-7 cells with HSV-TK/GCV treatment had obvious comet tails, and the expression level of DNA damage response active genes and proteins changed obviously in the HSV-TK/GCV treatment group,such as ATM, p53 and p27,but CyclinE and CDK2 did not change. CONCLUSION DNA damage on MCF-7 cells is resulted from HSV-TK/GCV in suicide gene therapy system through a p53-dependent signal pathway, causing cell cycle arrest and cell death.
Breast Neoplasms ; genetics ; pathology ; therapy ; DNA Damage ; Dependovirus ; genetics ; Female ; Ganciclovir ; metabolism ; pharmacology ; Gene Expression Regulation, Neoplastic ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Humans ; MCF-7 Cells ; Recombinant Fusion Proteins ; genetics ; metabolism ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics

OBJECTIVE: To investigate the mechanism and effect of diallyl disulfide (DADS) on the bystander effect of herpes simplex virus kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy system which was mediated by recombinant adeno-associated virus (rAAV) in lens epithelial cells. METHODS: Immunohistochemistry method was used to determine the distribution of connexin 43 (Cx43) protein in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene. Cx43 protein was measured and analyzed in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene that was induced by various DADS. Cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: DADS increased the Cx43 protein expression in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene, and especially in 50 μmol/L DADS. After combining with DADS, the bystander effect was significantly improved (P<0.05). CONCLUSION DADS can elevate the expression of Cx43 protein and enhance the bystander effect of HSV-tk/GCV suicide gene therapy system.
Adenoviridae ; genetics ; metabolism ; Allyl Compounds ; pharmacology ; Animals ; Antiviral Agents ; pharmacology ; Bystander Effect ; drug effects ; Cells, Cultured ; Connexin 43 ; metabolism ; Disulfides ; pharmacology ; Epithelial Cells ; metabolism ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Lens, Crystalline ; cytology ; drug effects ; metabolism ; Plant Oils ; Rabbits ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism

OBJECTIVETo evaluate the effect of adenovirus-mediated CD/TK double suicide gene system on tumor growth and cytokine levels in the tumor microenvironment in mice bearing transplanted colorectal cancer.

METHODSCT26 cells were implanted subcutaneously into 30 Balb/c mice, which were subsequently randomized into the control (n=15) and experimental group (n=15). After the tumor formation, CD/TK double suicide gene system was administered for tumor treatment, and the changes in the tumor volume, tumor inhibition rate, and levels of cytokines in the tumor microenvironment were investigated.

RESULTSCD/TK double suicide gene system resulted in a significant inhibition of the tumor growth and significantly increased levels of such cytokines as IL-2, IL-10, TNFalpha and IFNgamma in the tumor microenvironment.

CONCLUSIONCD/TK double suicide gene system produces significant tumor inhibition effect and causes obvious cytokine changes in the tumor microenvironment in mice bearing transplanted colorectal cancer.

Adenoviridae ; genetics ; metabolism ; Animals ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; therapy ; Cytokines ; metabolism ; Cytosine Deaminase ; genetics ; metabolism ; Female ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Green Fluorescent Proteins ; genetics ; metabolism ; Interleukin-2 ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; metabolism ; Thymidine Kinase ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism