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Objective:To explore the relationship between TK gene expression regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells.Method:The reformed reconstructed enhanced vector, pGL3-basic-EGFP-TK-hTRETp-CMV enhancer, and hTERT mono-promoter vector, pGL3-basic-EGFP-TK-hTRETp(as controls), were transfected into telomerase(+) nasopharyngeal carcinoma 5-8F cell lines,telomerase(+) human breast cancer MCF-7 cell lines and telomerase(-) normal vascular endothelium cell lines respectively. TK gene green fluorescent protein was observed by fluorescence microscope. The expression of TK gene mRNA was measured by the real-time fluorescent quantified PCR and the telomerase activity was determined by the method of TRAP argentation in maligment tumour cells pre- and post-transfected by enhanced vector . Meanwhile the relationship beteewn TK and telomerase was analyzed.Result:①A strong TK gene fluorescent show and TK mRNA expression were displayed after the enhanced suicide gene vector was transfected into nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which were more stronger than those of mono-promoter group,pGL3-basic-EGFP-TK-hTRETp,and ECV cells transfected by enhanced suicide gene vector. Meanwhile,real-time fluorescent quantified PCR showed that the A value of enhanced vector group was higher than that of controls. ②Telomerase activity after transfection of enhanced vector and GCV was lower than those before by the method of TRAP argentation in nasopharyngeal carcinoma cell lines,but no change in normal control cells after transfection of enhanced vector and GCV.③ After adding GCV, the obvious inhibitory effect of tumour cells growth induced by pGL3-basic-EGFP-TK-hTRETp-CMV enhancer were observed in nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which was higher than those of mono-promoter, pGL3-basic-EGFP-TK-hTRETp,pGL3-basic-EGFP3 and blank controls, but without inhibitory effect in ECV cells transfected by enhanced vector. Conclusion:TK gene expression is regulated by hTERT promoter and CMV enhancer, and then the telomerase activity is reduced and the cancer cells are specifically killed.But it is unclear how the telomerase are down-regulated by TK gene.
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Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. A recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system was construsted. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID_ 50 assay indicated that rPRV2 grows well on RK-13 cells. The LD_ 50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.
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Objective To study the killing action of adenovirus vector carrying HSV-tk(AdCMVtk) on Lewis cells.Methods The recombinant adenovirus carrying HSV-tk was constructed by homologous recombination techniques.The adenovirus transfected efficiency to Lewis cells was measured,the cells were transfected by adenovirus vector with LacZ gene,then stained with X-gal.The effect of AdCMVtk/GCV system on survival rate of Lewis cells was observed.Results The adenovirus transfected efficiency to Lewis cells was increased with the increasing of multiplicity of infection(MOI).100% transfected efficiency needed MOI 500.AdCMVtk/GCV system could kill Lewis cells.When the amount of adenovirus or the concentration of GCV was increased,the cell survival rate was decreased,but AdCMVtk or GCV alone could not kill Lewis cells.The killing action existed bystander effect,74% Lewis cells were died with only 20% Lewis cells transfected AdCMVtk.Conclusion AdCMVtk/GCV system is effective and safe on killing tumor cells.
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Objective To evaluate the suitability of the biodegradable microsphere encapsulation of adenovirus as a targeting vector for gene therapy of hepatocellular carcinoma. Methods Encapsulate the recombinant adenovirus in PLG poly (lactic/glycolic) copolymer by the solution evaporation method, the release test and the bioactivity of viruses incorporated in vitro were studied. Results More than 19.3% of adenovirus was encapsulated in PLG microspheres. The release test shows that the adenovirus was released for more than 200 h, 50% were shed within the first 100 h, and their activity was retained. Conclusion Recombinant adenovirus can be formulated in a polymer preparation of PLG with retention of bioactivity. It may be a valuable vector for the gene therapy of liver cancer.
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Objective To investigate the effects of spindle poisons (Vincristine, VCR and Colchicine, COL) on the tk gene of mouse lymphoma cells. Methods L5178Y cells were treated with VCR and COL at different concentrations. Determination of cytotoxicity, cell inoculation efficiency, relative survival rate, relative suspension growth rate and mutation frequency was performed. Results The relative survival rate and suspension growth rate of L5178Y cells decreased significantly with the increasing doses of VCR and COL. The mutation frequency of tk gene induced by VCR (0.5～2.5 ng/ml) and COL(10～50 ng/ml) was 1～3 times higher than that of spontaneous mutation frequency of L5178Y cells. Conclusion VCR and COL have obvious cytotoxic and mutagenic effects on tk gene in L5178Y cells.
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Objective:To investigate the effect of PEG-PEI/Fe3O4 Nano-magnetic fluid-TK on Apoptosis of liver cancer cells(HepG2)in vitro.Methods:Construcr ecombinant plasmid PEGFP-AFP-TK was constructed,which was delivered by nano-magnetic fuilds into AFP positive HepG2 cells and AFP negative SMMC7721 cells.The fluorescence was detected in order to evaluate the transfection rate 12h after transfection RT-PCR and Western blot were used to detect expression of HSV-TKgene at 48 h after transfection.MTT assay was used to evaluate the effect of HSV-TK on the proliferation of HepG2 cells,Flow cytometry was used to analyze the apoptosis of HepG2 cells.Results:Nano-Magnetic fluids delivered plasmid PEGFP-AFP-TK into HepG2 cells and HSV-TK gene was successfully expressed.The transfection eficacy of nano-magnetic fluids was superior than lipofectamine in HepG2 cells.RT-PCR and Western blot demonstrated that TK gene was expressed in HepG2 cells after being transfected with nano-magnetic fluids/PEGFP-AFP-TK.MTT and flow cytometry showed HSV-TK gene exerts a cell-killing effect.Conclusions:Nano-Magnetic fluids could successfully deliver PEGFP-AFP-TKinto HepG2 cells and induce expression of HSV-TK,it may become a promising gene vector for liver cancer gene therapy.AFP enhancer can specifically enhance the expression of target gene within the cell with positive AFP.
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Objective To investigate the selective killing effect of adenoviral mediated herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene system controlled by human telomerase reverse transcriptase (hTERT) promoter on bladder cancer cells in vitro. Methods Bladder cancer cell line 253J and human fibroblast cell line MRC-5 were transfected by the recombinant adenovirus of different multiplicities of infection (MOI),and the infection rate was measured by observing the expression of enhanced green fluorescent protein (EGFP) under the fluorescent microscopy.Ad-hTERT-HSV/tk and Ad-CMV-HSV/tk were transduced into 253J and MRC-5,followed by GCV treatment. The relative survival rate of cells in presence of prodrug GCV was measured with MTT method. Results Recombinant adenovirus Ad-hTERT-EGFP could selectively infect 253J cells,with the infection rate associated with the increasing MOI of recombinant adenovirus (P
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Objective To investigate and compare the killing effects of different prodrugs combined with suicide gene HSV-tk on laryngocacinoma cell, Hep-2 in vitro. Methods Retroviral expressing vector pL(tk)SN was constructed by recombinant DNA technology. Hep-2 cells were infected by the recombinant retrovirus. The positive cloning was obtained after G418 selection and were termed Hep/tk. The integration and expression of tk gene in Hep-2 cells were identified by RT-PCR and Southern blot. The growth state and prodrugs killing effect of tk gene modified cells were used to investigate the expression of tk gene and antitumour effect on Hep-2 cells. Results RT-PCR and Southern blot analysis confirmed the integration and expression of tk gene in Hep-2 cells. There was no significant difference in cell proliferation between the Hep/tk and Hep-2. After the treatment of GCV,the Hep/tk showed high sensitivity to GCV and bystander effects were observed siginificantly in vitro. However the efficiency of another two prodrugs ACV and BVdU was lower than that of GCV. Both tk-positive and tk-negative Hep-2 cells were relatively insensitive to ACV and BVdU.Treatment of tk gene modified cells mixed with different proportion parental cells shoewd obviously bystander effects.Conclusions The laryngocarcinoma cells Hep-2 have sensitive to HSV-tk/GCV system and have significant bystander effects, which might have therapeutic potential value for laryngocarcinoma.
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PURPOSE: Gastric cancer is the most common malignancy in Korea. Although treatment such as surgery, chemotherapy, and immunotherapy has greatly improved, the mortality rate of gastic cancer is still high, A new therapeutic trial is necessary to improve the cure rate of gastric cancer. Therefore we investigated the pre-clinical significance of HSV-tk gene therapy using retroviral vector for gastric cancer cell lines. MATERIALS AND METHODS: LNC/HSV-tk retroviral vector and PA317/LNC/HSV-tk producer cell line were constructed. HSV-tk gene transduction and expression were detected by PCR. An in vitro ganciclovir(GCV) sensitivity test was performed by MTT assay. To evaluate in vivo GCV sensitivity, GCV was intraperitoneally injected after tumor formation in the nude mice. Bystander effect was observed in vitro MTT assay using YCC- S-2 cell line and in vivo using N87 and YCC-S-2 cell lines. RESULTS: The in vitro GCV sensitivity test showed that the growth inhibition was 30~32% with 0.5 uM GCV and 52~77% with 500 uM GCV in the HSV-tk transduced cell line in comparison with 0- 5% with 0.5 and 500 uM GCV in the parent cell line. The in vivo GCV administration showed that the tumors induced by HSV-tk transduced N87 cell line and YCC-S-2 cell line decreased completely, while the tumors with the parent cell lines continued to grow in nude mice. We observed no tumor cells in tissue specimen of the tumor induced by the N87/HSV-tk cell line after. GCV administration. In vitro and in vivo bystander effects were observed in HSV-tk/GCV system due to the resultant cell death exceeding the proportion of HSV-tk transduced cells in the mixtures of HSV-tk transduced and parent cells. CONCLUSION: HSV-tk transduced gastric cancer cell lines showed sensitivity to GCV and a bystander effect was observed. These results suggested that HSV-tk/GCV system should be evaluated in the clinical settings.
Subject(s)
Animals , Humans , Mice , Bystander Effect , Cell Death , Cell Line , Drug Therapy , Ganciclovir , Genetic Therapy , Herpes Simplex , Immunotherapy , Korea , Mice, Nude , Mortality , Parents , Polymerase Chain Reaction , Stomach Neoplasms , Thymidine Kinase , Thymidine , ZidovudineABSTRACT
BACKGROUND: Replication of defective adenoviral vectors can be used for gene transfer into a wide spectrum of replicating and nonreplicating cells. Accordingly, selective introduction of genes encoding for susceptibility to nontoxic drugs into proliferating tumor may be used to treat cancer. We investigated the efficacy of in vitro transduction of neuroblastoma cell with the herpes simplex virus thymidine kinase gene followed by administration of the antiviral drug ganciclovir. METHODS: The recombinant adenoviral vector ADV/TK carrying the HSV-TK gene, under the control of the promoter from Rous sarcoma virus long term terminal repeat was constructed. And 1 x 10(4) Neuro 2a cells were plated in 96 well cultured plates and infected with ADV/TK at multiplicity of infection of 0, 1, 10, and 100. Twenty-four hours later, the infected cells were treated with PBS or ganciclovir at a concentration of 10 g/ml. After 48hr, the surviving cells in 96 well plates were determined by MTT assay. RESULTS: After infection in vitro with ADV/TK at moi of 0, 1, 10, 100 and subsequent ganciclovir treatment, the percent survival rate of 1 x 10(4) Neuro 2a cells were 105%, 32%, 25%, and 15%. But the survival rate of 1 x 10(4) Neuro 2a cells with PBS treatment were 100%, 92%, 105%, 103%. CONCLUSION: We concluded that in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir is very effective.
Subject(s)
Cell Line , Ganciclovir , Neuroblastoma , Phosphotransferases , Rous sarcoma virus , Simplexvirus , Survival Rate , Terminal Repeat Sequences , Thymidine KinaseABSTRACT
Objective: To determine if fusion genes of HSV-tk gene and cytokine gene have synergy on the cell killing of the Hep-2 human laryngeal carcinoma cell line in vitro. Methods: Different fusion genes expressing vectors PL(TI) SN, PL(TT)SN and PL(TK)SN were generated by recombinant DNA technology. Hep-2 was infected by the recombinant retrovirus. The positive clones were obtained after G418 selection and were termed Hep/TI, Hep/TT and Hep/TK respectively. The integration and expression of fusion genes in Hep-2 cells were identified by RT-PCR and Southern blot. The growth state and GCV killing effect of fusion genes modified cells were used to investigate the expression of fusion genes and antitumour effect on Hep-2 cells. Results: RT-PCR and Southern blot analysis confirmed the integration and expression of fusion genes in Hep-2 cells. There was no significant difference in cell proliferation between the Hep/TI and Hep/ TK.but the growth of Hep/TT was restrained. After the treatment of GCV the Hep/TI, Hep/TT and Hep/TK all showed high sensitivity to GCV. The killing effect of GCV on Hep/TT was the most siginificant and bystander effects were observed siginificantly in vitro. Conclusion: The fusion genes of HSV-tk and cytokine gene have synergistic effects on killing Hep-2 cell after treatment of GCV in vitro,which might have therapeutic potentials for laryngocarcinoma.