Preclinical study of suicide gene as a safety switch to control CAR-T cell cytotoxicity / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate whether AP1903, a small-molecule chemical inducer, can terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vivo and in vitro. Methods: CD19CAR-T cells over-expressing iCasp9 (iCasp9-CD19CAR-T) were constructed and co-incubated with AP1903. Then, the cell phenotype and apoptosis were detected by Flow cytometry, and the iCasp9/CID suicide gene system was verified on K562 and T cells, respectively. The cytotoxicity of iCasp9-CD19CAR-T cells was detected in vivo (survival rate of NCG mice bearing Raji cell transplanted xenograft) and in vitro (cell killing function was detected by Flow cytometry) under the administration of AP1903. Results: Compared with CD19CAR-T cells, iCasp9-CD19CAR-T cells showed in significant difference in proliferation, phenotype and cytotoxicity both in vitro and in vivo (all P>0.05). At 2 h after AP1903 administration, the apoptosis rates of K562 and T cells co-expressing iCasp9 and CD19CAR were (33.8±0.9)% and (27.95±0.35)%, respectively; and at 24 h after AP1903 administration, the apoptosis rates reached 100% in both cell lines. The in vitro cytotoxicity of iCasp9-CD19CAR-T cells induced by AP1903 was significantly lower than that without AP1903 treatment (P<0.01); the 60-day survival rate of mice bearing Raji cell transplanted tumor treated with AP1903-induced iCasp9-CD19CAR-T cells was also significantly lower than those treated with iCasp9-CD19CAR-T cells alone (P<0.01). Conclusion: AP1903 can effectively terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vitro and in vivo.

Combination of TMPRSS2 promoter regulation of CD-5-FC gene system and phytochemical drugs effect prostate cancer 22RV1 in vitro / 中华泌尿外科杂志

Objective To explore the effects of TMPRSS2 promoted suicide gene CD-5-FC combined with phytochemicals on the proliferation and apoptosis of prostate cancer (PCa) specific cell-line 22RV1.Methods From March 2016 to October 2016 TMPRSS2-VISA-CD/UPRT'vector was used to deliver pro-drug 5-FC (5-Fluorocytosine) in to PCa specific cell-line 22RV1.Transfection effect was verified by Western-blotting.5-FC,5-FU (5-Fluorouracil) and four phytochemicals epigallocatechin gallate (ECGC),Genistein,Daidzein,and Equol were selected in this study.MTS assay was performed to select dosages,which can kill 10％-40％ of 22RV1 cells,from their pre-set drug concentrations,respectively.According to their select concentrations,either 5-FC plasmid or 5-FU in combination with one of four phytochemicals were added in 22RV1 cells in orders,and then cultured them together.Besides,these four phytochemicals were added in 22RV1 cells individually regarding their selecting concentrations and cultured at the same time.Cell viability was detected by MTS assay on 24hrs,48hrs and 72hrs.Comparing the cell killing rates between the combination groups and each single drug groups on cell-line 22RV1.Results TMPRSS2-VISA-CD/UPRT'plasmid with 5-FC was transfected on PCa specific cell-line 22RV1 successfully.The most obvious transfection occurred on 48hrs.The selected concentration of the pro-drug 5-FC were 83.32 μmol/L and 833.20 μmol/L,5-FU were 76.87 mol/L and 768.70 μmol/L.The concentrations of four phytochemical agents were 2.00 μmol/L,10.00 μmol/L and 20.00 μmol/L.In term of the cell killing numbers,results showed that the combination treatment of 83.32 μmol/L 5-FC/ 76.87μmol/L 5-FU and 10.00 μmol/L/20.00 μmol/L four phytochemicals compared with 10.00 μmol/L/20.00 μmol/L single treatment of four phytochemicals,respectively,there was no significant difference (P ＞0.05),while the combination treatment of 833.20 μmol/L 5-FC/768.70 μ mol/L 5-FU and 2.00 μmol/L/10.00 μmol/L four phytochemicals compared with 2.00 μ mol/L/10.00 μmol/L single treatment of four phytochemicals,respectively,there was a significant difference (P ＜ 0.05).In addition,this study proved that 83.32 μmol/L 5-FC and 76.87 μmol/L 5-FU,833.20 μmol/L 5-FC and 768.70 μmol/L 5-FU reached the same cell killing effect,and there was no statistical difference (P ＞ 0.05).Thus the suicide gene transfection was successful,and its function was the same as the effect of cytotoxic drugs.Conclusions This study has proved that this plasmid with 5-FC suicide gene system can be successfully and efficiently transfected into PCa cells 22RV1.As the pro-drug of 5-FU,5-FC got similar treatment effect with 5-FU,and inhibited cell proliferation.There was no synergistic reaction of enhancing cell apoptosis after combined phytochemical drugs EGCG,Genistein,Daidzen,Equol with suicide gene therapy on 22RV1 cell-line.

Apoptosis induced by transferring cytosine deaminase suicide gene into kinds of cancer cells with novel polyamidoamine dendrimers / 药学学报

This study demonstrates efficacy of a novel polyamidoamine dendrimers (PAMAM dendrimers) with pentaerythritol derivatives as the core (G5 PD dendrimer) in deliver of the cytosine deaminase (CD) gene and EGFP gene fusion plasmid into different tumor cell lines to induce apoptosis. The physical and chemical properties of G5 PD dendrimers in terms of DNA complexation, particulate properties and transfection efficiencies were investigated and compared with commercial gene vectors PEI 25 kDa. The optimum ratio of G5 PD dendrimer complexed with plasmid DNA was 0.2:1, and the particle size of the complex was (100±5) nm. Compared with the commercial gene carriers PEI, G5 PD dendrimer exhibited a higher transfection efficiency at the weight ratio of 1:1 in three different cell lines, given the fact that PEI are different from PAMAM dendrimers in terms of molecular structure. Furthermore, the cytotoxicity assays of the cell lines transfected with G5 PD dendrimer/pCD-EGFP-N1 followed by exposure to various concentrations of 5-fluorocytosine (5-FC) also showed that the transfected cell lines could generate a very low amount of 5-FC to 5-fluorouracil (5-FU) in a short period of time, which indicating the high expression level of CD gene in the cell line. These results indicate that the CD/5FC system of G5 PD dendrimer has an excellent efficacy in gene delivery.

Progression of recombinant adenovirus mediated herpes simplex virus thymidine kinase for the treatment of liver cancer / 中华消化外科杂志

Liver cancer is one of the most common malignant tumors in China.The efficacy of traditional treatment for liver cancer is unsatisfactory,and the prognosis of the patients is poor.In recent 10 years,with the development of the molecular biological techniques,genetic therapy has become a new and promising approach for liver cancer.Of which,adenovirus mediated herpes simplex virus thymidine kinase (ADV-tk) for the treatment of liver cancer is widely applied.The enzyme secreted by ADV-tk transformed the prodrug gancyclovir (GCV) to the cytotoxic agents and thus to kill the liver cancer cells.The results of multiple animal and clinical experiments showed that ADV-tk/GCV is effective for the treatment of liver cancer.In this article,the recent progress of ADV-tk/GCV in the treatment of liver cancer was reviewed.

An overview of gene therapy in head and neck cancer.

Gene therapy is a new treatment modality in which new gene is introduced or existing gene is manipulated to cause cancer cell death or slow the growth of the tumor. In this review, we have discussed the different treatment approaches for cancer gene therapy; gene addition therapy, immunotherapy, gene therapy using oncolytic viruses, antisense ribonucleic acid (RNA) and RNA interference‑based gene therapy. Clinical trials to date in head and neck cancer have shown evidence of gene transduction and expression, mediation of apoptosis and clinical response including pathological complete responses. The objective of this article is to provide an overview of the current available gene therapies for head and neck cancer.

Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells

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.

Updates on current advances in gene therapy / Actualizaciones acerca de los adelantos presentes en terapia genética

Gene therapy is the attempt to treat diseases by means of genetic manipulation. Numerous challenges remain to be overcome before it becomes available as a safe and effective treatment option. Retroviruses and adenoviruses are among the most commonly used viral vectors in trials. The retrovirus introduces the gene it carries into the target cell genome while the adenovirus introduces the gene into the target cell nucleus without incorporating it into the target cell genome. Other viral vectors such as adenoassociated viruses, pseudotyped viruses and herpes simplex viruses, are also gaining popularity. Proposed nonviral methods for gene transfer include physical methods and the employment of chemical vectors (lipoplexes, polyplexes and inorganic nanoparticles). Recent studies have investigated potential applications of gene therapy in correcting genetic diseases, treating malignant disorders and for treatment of other diseases. Trials on gene therapy for SCID and Leber's congenital amaurosis have achieved considerable success, but the widely publicized adverse reaction in Xlinked SCID patient receiving gene therapy raised concerns for safety profile of gene therapy. For that, several methods of improving safety and efficacy of gene therapy have been proposed. At present, the three main gene therapy strategies for treatment of cancer are application to oncolytic viruses, suicidegene therapy and genebased immunotherapy. Gendicine, the first approved anticancer drugs based on the use of gene therapy principle, is based on the use of oncolytic viruses. More evidence for wider clinical applications of gene therapy are expected as more gene therapy studies progress from the preclinical phase to clinical trial.

La terapia genética es el intento de tratar enfermedades por medio de la manipulación genética. Quedan aún numerosos retos que superar antes de que esté tipo de tratamiento se encuentre disponible como una opción segura y eficaz. Los retrovirus y los adenovirus se hallan entre los vectores virales más comúnmente utilizados en ensayos: el retrovirus introduce el gen - del cual es portador - en el genoma de la célula de destino, mientras el adenovirus introduce el gen en el núcleo de la célula de destino sin incorporarlo al genoma de la célula de destino. Otros vectores virales tales como los virus adenoasociados, los virus pseudotipados, y los virus del herpe simple, también están ganando popularidad. Los métodos no virales propuestos para la transferencia de genes incluyen tanto métodos físicos como el empleo de vectores químicos (lipoplexes, polisomas y nanopartículas inorgánicas). Estudios recientes han investigado las aplicaciones potenciales de la terapia genética en la corrección de las enfermedades genéticas, el tratamiento de los trastornos malignos y para el tratamiento de otras enfermedades. Los ensayos de terapia genética para SCID y la amaurosis congénita de Leber han logrado un éxito considerable, pero la reacción adversa ampliamente divulgada en el caso de los pacientes con SCID ligada al cromosoma, que recibían terapia génica, causó preocupación en cuanto al perfil de seguridad de la terapia génica. Por esa razón, se han propuesto varios métodos para mejorar la seguridad y la eficacia de la terapia génica. En la actualidad, las tres estrategias principales de terapia de genes para el tratamiento del cáncer son la aplicación de virus oncolíticos, la terapia con gen suicida, y la inmunoterapia genética. La gendicina, el primer medicamento anticancerígeno aprobado, basado en el uso del principio de la terapia génica, se basa en el uso de virus oncolíticos. Se esperan más evidencias a favor de aplicaciones clínicas más amplias de la terapia génica, a medida que más estudios de terapia génica progresan de la fase preclínica a la fase de ensayo clínico.

Suicide gene driven by human alpha fetoprotein enhancer kills hepatocellular carcinoma cells / 中华消化外科杂志

Objective To investigate the effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GGV)system driven by human alpha fetoprotein(AFP)enhancer on hepatocellular carcinoma(HCC)cells in vitro and in vivo.Methods HCC-specific eukarotypic expression vector carrying suicide gene driven by AFP enhancer(pAFP-cDNA3.1-TK)was constructed.The plasmid was trasfected to AFP-positive HepG2 cells and AFP-negative SMMC7721 cells by liposomes.Protein and mRNA expressions of TK were detected by RT-PCR or Western blot.The survival rates of HCC cells were detected by methyl thiazolyl tetrazolium assay.The effects of GGV on the in vitro proliferation,survival and apoptosis of HCC cells were observed,and the inhibitive effect of GGV on the survival of HCC cells in vivo was also detected.All data were analyzed by using the t test.Results The pAFP-cDNA3.1-TK was successfully constructed and transfected to the HCC cells.The protein and mRNA expressions of TK were detected in AFP-positive HepG2 cells.GGV dose-and time-dependently inhibited the growth and induced the apoptosis of HepG2 cells in vitro,but it had no effect on SMMC7721 cells.No protein or mRNA expression of TK was detected in the SMMC7721 cells.There was a significant difference on the inhibitory effects of GGV on HepG2 cells and SMMC7721 cells(t =2.58,2.73,3.12,P ＜0.05).GGV specifically inhibited the growth of AFP-positive HepG2 cells,and the inhibition rate was 46%;the growth of AFP-negative SMMC7721 cells was not influenced by GGV.There was a significant difference in the inhibitive effect of GGV on the growth of HepG2 cells and SMMC7721 cells(t = 3.36,P ＜ 0.05).Conclusion HSV-TK/GGV systemdriven by human APF enhancer kills APF-positive HCC cells and inhibits the growth of HCC cells.

Effect of suicide gene therapy in combination with immunotherapy on antitumour immune response & tumour regression in a xenograft mouse model for head & neck squamous cell carcinoma.

Background & objectives: Prodrug activation strategy as well as immunotherapy have been widely used for cancer gene therapy. In the present study, using a head and neck squamous cell carcinoma (HNSCC) xenograft nude mouse model, we have investigated whether the two therapies in combination could improve tumour cell kill. We also investigated induction of immune effector cells viz., NK (DX5+) and DC (CD11c+) in vivo, post-combination gene therapy. Methods: A retroviral vector producing cell line (PLTK47.1 VPC) carrying Herpes simplex virus thymidine kinase gene (HSVtk) was used for intratumoural injection into NT8e xenograft tumours followed by the prodrug ganciclovir (GCV). IL-2 plasmid DNA was injected intramuscularly. Immune cells were analyzed by flow-cytometry. Non parametric ANOVA was performed with Kruskal Wallis test. Results: IL-2 could induce proliferation of both NK cells (DX5+) and dendritic cells (CD11c+) in vivo. Apoptosis was higher in combination therapy group as compared to HSVtk/GCV alone or IL-2 alone and was mediated through caspase-3 dependent pathway. Significant reduction in tumour volume was seen in all 3 treatment arms as compared to controls. Interpretation & conclusions: Combination of suicide gene therapy and immunotherapy leads to successful tumour regression in a HNSCC xenograft mouse model. Immunotherapy could help in a systemic long lived anti-tumour immune response which would prove powerful for the treatment of metastatic cancers, and also for minimal residual disease. The results of this study may form the basis for Phase 1 clinical trials.

Treatment of human pancreatic cancer with adenovirus-mediated fusion gene system driven by KDR promoter in nude mice / 中华肝胆外科杂志

Objective To investigate the curative effect of the adenovirus-mediated fusion gene system driven by KDR promoter (AdKDR-CDglyTK) on a model of pancreatic cancer. Methods By using transplantation of the cultivated cells, human pancreatic cell line Capan-2 was injected subcutaneously on the back of nude mice to establish the animal model of the pancreatic cancer. Twenty nude mice were divided randomly and equally into four groups. The mice in group Ⅰ were injected with AdKDR-CDglyTK and 5-FC/GCV, those in group Ⅱ were injected with 5-FC/GCV, those in group Ⅲwere injected with AdKDR-CDglyTK and those in group Ⅳ received no any injection. AdKDR-CDglyTK was injected directly into the tumor and 5-FC/GCV was given by intraperitoneal injection. The observing parameters included common status, tumor bulk, tumor weight, inhibition rate of tumor growth, pathology, immunohistochemistry and treatment effect in each group. Electron microscopy was performed to observe the pathological changes of cells. The apoptotic cells in tumor were detected using the TUNEL assay. The expression of CDglyTK in tumors from each group was examined by RT-PCR. Results Tumor growth was dramatically inhibited in group Ⅰ. Tumor growth has no significant difference among groupⅡ , group Ⅲ and group Ⅳ. The apoptotic rate (34.20±4.60)% was significantly increased in group Ⅰ (F= 243. 22, P= 0. 00) and it had no significant difference among groupⅡ , group Ⅲ and group Ⅳ (P＞0.05). Conclusion AdKDR-CDglyTK with 5-FC/GCV can obviously inhibit the growth of human KDR-expressing pancreatic cell line Capan-2 and induce the cell apoptosis in vivo. The probable molecular mechanism lies in the facts that the system can cause a decline in the level of Bcl-2.

Radiobiology effects of radiation-induced horseradish peroxidase/indole-3-acetic suicide gene expression in lung cancer cells / 中华放射肿瘤学杂志

Objective To detect specific cell killing effect of radiation combined with horseradish peroxidase (HRP)/indole-3-acetic (IAA) suicide gene therapy controlled by a novel radio-inducible and cancer-specific chimeric gene promoter in lung cancer. Methods We constructed a plasmid expressing HRP enzyme under the control of chimeric human telomerase reverse transcriptase (hTERT) promoter carrying 6 CArG elements, a plasmid expressing HRP enzyme under the control of hTERT promoter carrying single CArG element, and two control plasmids, which named pE6-hTERT-HRP, phTERT-HRP, pControl-HRP, and pControl-luc, respectively. After radiation, the proliferation inhibition and apoptosis induction effect of each type of plasmid in lung cancer cells (A549, SPC-A1) and normal lung cells (hEL) was detected by cell counting and Annexin V-FITC staining. The change of radiosensitivity of lung cancer cells with plasmid system was also detected by clonogenic assays. Results After a single dose radiation of 6 Gy,the average proliferation inhibition rates of pE6-hTERT-HRP, phTERT-HRP, pControl-HRP, and pControl-luc systems were 72. 92% ,40.60% , 51.00% and 25.19% (F= 67.31 , P< 0.01) in A549 cells ,64.63%,30.02%,48.23% and 23.16% (F=64.94, P< 0.01) in SPC-A1 cells, and 20.81%,18.05%, 44.20% and 18.32% (F=52. 19,P<0.01) in normal hEL cells, respectively. The average early apoptosis rates of these four plasmid systems were 36. 63%, 22. 30%, 24. 33% and 12. 53% (F =50. 99,P <0. 01) in A549 cells, 33.73%, 17. 37%, 22. 43% and 11.20% (F = 20. 76, P < 0. 01) in SPC-A1 cells, and 13.53 %, 12. 5%, 21.93% and 12. 16% (F = 15. 08, P < 0. 01) in normal hEL cells,respectively. The sensitizing enhancement ratios of the four plasmid systems were 3.45, 2. 29, 3.05 and 1.21 in A549 cells, while 2. 68, 2. 15, 3.05 and 1.21 in SPC-A1 cells, respectively. Conclusions The new suicide gene system controlled by chimeric promoter may provide a novel therapeutic modality for lung cancer.

Killing effects of PNP-CD chimeric suicide gene vector on HCC cells / 中华肝胆外科杂志

Objective To investigate the cytotoxic effects and mechanism of PNP-CD chimeric gene vector originated from PNP/MeP-dR system on HCC cells. Methods The fusion suicide gene PNP-CD obtained by site directed mutagenesis technique was subcloned into pcDNA3.0 to construct a eukaryotic expression vector containing a chimeric gene, pcDNA3.0/ PNP-CD. After being identified by recombinant enzyme, PCR and subsequent sequencing, it was transfected into HepG2 cells by liposome-mediation method. The G418-resistant cellular clone with stable transfection of pcDNA3.0/PNP-CD, HepG2/PNP-CD was established by selection. The expression of PNP-CD gene was also verified by RT-PCR and Western blotting. The curve of cellular growth was assayed by Trypan blue exclusion. The cellular sensitivity of HepG2/PNP-CD to its specific prodrugs and its bystander effects were also assayed by MTT method. Results The chimeric gene, PNP-CD, was inserted into pcDNA3.0 correctly, and the stable expression of pcDNA3.0/PNP-CD in HepG2 cells was confirmed.This cellular clone was highly sensitive to its corresponding prodrugs. It was indicated that its bystander effects with the synergetic treatment of its specific prodrugs were substantially higher than those caused by the same vector with the administration of only a single prodrug, MeP-dR. Conclusion The bi-functional fusion suicide gene vector, pcDNA3.0/PNP-CD, yields powerful cytotoxic effects on HCC cells in the presence of the synergetic treatment of its specific prodrugs, which would be a high-performance therapeutic vector in gene therapy for liver cancer.

Current progress in bystander effect of suicide gene therapy / 国际外科学杂志

Nowadays, tumor is one of the most challenging human health diseases, which oblige human tirelessly to studied for the tumor.Gene therapy is a promising treatment in oncotherapies. Suicide gene therapy is the most widely researched among gene therapies. At the same time, bystander effect take important role in the mechanism of suicide gene therapy. Therefore, more researchers devote themselves to studyinghow enhance the bystander effect in order to improve the effect of suicide gene therapy. This article reviewed in short how to augment bystander effect of suicide gene therapy against cancer.

Study on the relationship between TK gene regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells / 临床耳鼻咽喉头颈外科杂志

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.

Construction and application of conditionally replicative adenovirus for selective cytotoxicity in CEA positive colorectal cancer cells / 上海第二医科大学学报

Objective To construct a new conditionally replicative adenovirus (CRAds) targeting carcinoembryonic antigen (CEA) positive colorectal cancer cells. Methods The DNA fragment of the CEA gene promoter was amplified through PCR and cloned into the vector carrying fusion reporter gene EGFP-Luc to construct expression plasmid pCEA-EGFPLuc. The constructed plasmid pCEA-EGFPLuc was transfected into CEA positive and negative cells by liposome. The activity of CEA gene promoter was evaluated by detecting the expression of EGFP and luciferase activity. The conditionally replicative adenovirus Ad.CEA-E1A/CMV-TK carrying suicide gene HSVtk was constructed, in which the E1A gene was controlled under CEA promoter. CEA positive(Lovo and SW620)and negative tumor cells(HeLa) were infected with Ad.CEA-E1A/ CMV-TK. The selective cytotoxicity of Ad.CEA-E1A/CMV-TK and the synergistic effect of the virus with GCV in CEA positive tumor cells were evaluated by the expression of E1A, cytopathic effect and cell survival rate. Results CEA promoter possesses a good specificity as well as high activity. The expression of E1A only presented in CEA positive tumor cells. After infection with Ad. CEA-E1A/CMV-TK, the cell survival rates of Lovo and SW620 were (36.72±2.49)% and (39.82±4.76)%, respectively, significantly lower than that of Hela[(87.44±2.76)%1 (P<0.01). When combined with GCV, Ad.CEA-E1A/CMV-TK had better oncolytic effect on Lovo and SW620 cells, with cell survival rates of (17.26±3.65) % and (23.93±5.40) %, respectively, significantly lower than those without GCV[(36.72±2.49) % and (39.82±4.76) %, respectively] (P<0.01). Conclusion Ad. CEA-E1A/CMV-TK under the control of CEA promoter has selective cytotoxic effect on CEA positive colorectal cancer cells, and the effect can be enhanced when combined with GCV.

Inhibitory effect of mutant cytosine deaminase D314A against human colon cancer cells / 中国肿瘤生物治疗杂志

Objective:To construct a mutant D314A of Escherichia coli cytosine deaminase (EC-CD, substitution of an alanine (A) for the aspartic acid (D) at position 314 of cytosine deaminase) and investigate its antitumor effect. Methods: Eukaryotic expression plasmid containing EC-CD gene (pcDNA3.1-CD~(wt)) was constructed, and the mutant pcDNA3.1-CD~(D314A) plasmid, with aspartic acid (D) at position 314 of EC-CD gene substituted by alanine (A) (EC-CD~(D314A)), was established by site-directed mutation. EC-CD~(wt) and EC-CD~(D314A) were transfected into human colon cancer cell line LoVo via Lipofectamine~(tm) 2000, and positive LoVo-CD~(wt) and LoVo-CD~(D314A) cells stably expressing corresponding genes were selected by G418. The cytotoxicity and bystander effects of EC-CD and EC-CD~(D314A) genes on LoVo cells were e-valuated by MTT assay. Results: The mutant D314A was confirmed by sequence analysis. EC-CD and EC-CD~(D314A) mRNA were expressed after transfected into LoVo cells. The IC_(50) of Lovo-CD~(D314A) cells was (85.13±0.60) mmol/L, which was significantly lower than that of LoVo-CD~(wt) cells ([689.76±0.45] μmol/L, P=0.000). Bystander effect assay showed that, when at the ratio of 30%, the survival rates of LoVo-CD~(wt) cells and Lovo-CD~(D314A) cells were (48.5±0.49)% and (17.3±0.40) % (P = 0.000), respectively. Conclusion: Mutatant EC-CD gene (EC-CD~(D314A)) has a significantly in-creased antitumor effect on LoVo cells compared with wild type EG-CD gene, and it may become a new candidate gene for tumor gene therapy.

Construction of pEGFP-N1-Fcy::Fur recombinant plasmid and observation of its expression in ovarian cancer cells / 重庆医科大学学报

Objective:To construct a EGFP(Enhanced green fluorescent protein)-labled euk-aryotic expression plasmid of Fcy::Fur suicide gene and to detect its expression in SKOV3 cell line. Methods:With the technology of gene re-arrangement,Fcy::Fur gene in pORF-Fcy:Fur plasmid was subcloned into pEGFP-N1 vector,with its correctness evaluated by the means of r-estriction enzyme analysis and sequencing.It was transfected into SKOV3 cells with lipofectin,the transient expression of GFP was observed under flu- orescence microscope after 24 hours and detected by Western blot. Results:Correct construction of pEGFP-N1-Fcy::Fur was identi- fied by methods of restriction enzyme analysis and nucleotide sequence determination.A total of 60% transfe-cted cells emitted out green fluorescence under fluorescent microscope after 24 h after transfecti-.on. Fcy::Fur gene expressed by the transfected cells were testified by Western blot. Conclusion:The recombinant eukaryotic expression vectors have been constructed successfully and effec- tive-ly expressed in ovarian cancer cells,which may provide an experimental basis for gene therapy of ovarian cancer.

Study on AdCD-UPRT suicide gene mediated on lung cancer A_(549) in vitro / 重庆医科大学学报

Objective:To study combination AdCD-UPRT suicide gene mediated on lung cancer A549 in vitro. Methods:The recombinant AdCD-UPRT adenovirus was packaged and generated,and the title of virus was measured. The efficiency of transfecting target cells was detected,the toxicity of virus was measured,and the expression of CD-UPRT gene in A549 cells was assayed. The cytotoxicity and the bystander effect on A549 cells were measured by MTT assays respectively. The apoptosis was analyzed by AO-EB fluorescent staining,transmission electron microscope and flow cytometry. Results:The combination AdCD-UPRT had obviously growth inhibition effect on A549 cell and bystander effect. The cells were blocked in S phase and induced into apoptosis. Conclusion:The combination AdCD-UPRT suicide gene had cytotoxicity on lung cancer A549 in vitro.

pAdKDR-tk and pAdCMV suicide gene system cytotoxicity and specificity in vitro / 肿瘤研究与临床

Objective In order to identify cytotoxicity and specificity of the suicide gene systems which vascular endothelial growth factor receptor 2(KDR) and CMV were used as promoters respectively. Methods Two cell lines, HUVEC and CNE-2, one expresses KDR highly,another doesn't, were used as target cells for identifying the cytotoxicity and specificity of recombinant adenoviral vectors with herpes simplex virus thymidine kinase (HSV-tk) gene expression driven by KDR promoter and CMV promoter respectively (pAdKDR-tk and pAdCMV-tk) in Vitro by adding GCV. Results HUVEC and CNE-2 showed similarly sensitivity to GCV when they were transfected by pAdCMV-tk. However, They shown different sensitivity to GCV when they were transfected by pAdKDR-tk. When HUVEC was transfected in high MOI (MOI=100) and concentration of GCV was 50 ?g/ml, The cell growth rate decreased to (23.12?4.90)% .On the contrary, CNE-2 cells growth rate remained as high as (70.46 ?3.27)%. Conclusion Two kinds of cell line CNE-2 and HUVEC can be killed by pAdCMV-tk suicide gene system, but pAdKDR-tk suicide gene system only can kill HUVEC cell line. The result implied that pAdKDR-tk could kill the cells that express KDR specifitly.

Inhibition by Berberine and Cytosine Deaminase Enzyme Suicide Gene on Rectum Cancer Cells / 医学研究杂志

Objective To investigate the killing effect by berberine and cytosine deaminase enzyme suicide gene on rectum cancer cells in vitro.Methods Human rectum cancer HR-8348 cells were cultured,then transfected by recombinant adenovirus suicide gene the survivial percentage of which was examined by MTT method.Results Recon of pcDNA3.1(+)-CD was constructed successfully.The CD gene sequence analysis indicated th a+ recon pcDNA3.1(+)-CD contain intact gene.RT-PCR showed CD gene expressed stably in the HR-8348 cells,which is sensitive to 5-FC above the density of 100?g/ml.Elevated along with the medicine density with response time lengthening,its damaging effect enhanced.However for the density above 5-FC 250g/ml,the change of cell killing rate was not apparent.when by 0.1,0.3,3.0,30.0?m density berberine unites the 5-FC on rectum cancer of the intestines Respectively,the cell inhibitory action elevates along with density rises,but 3.0?m,30?m the inhibition change was not apparent.Under 250?g/ml of 5-FC,0.1,0.3,3.0,30.0?m of berberine united the 5-FC The HR-8348 cells growth suppression rate were:27.7%,42.4%,52.3%,56.3% respectively.Conclusion Inhibition function to human rectum cancer HR-8348 cells can be strengthened by berberine combined 5-FC.