OBJECTIVETo construct a replication-defective adenovirus containing TK gene and investigate the killing effects of TK gene against human liver cancer cells SMMC-7721.

METHODSThe recombinant adenovirus ADV-TK was constructed using homologous recombination in the cells. SMMC-7721 cells transfected with recombined adenovirus were exposed to GCV, and the cell viability was measured by MTT assays.

RESULTSThe recombinant adenovirus containing TK gene was successfully constructed. Transfection by the recombinant adenovirus ADV-TK and GCV exposure significantly suppressed the growth of SMMC-7721 cells.

CONCLUSIONA replication-defective adenovirus containing TK gene has been successfully constructed, and in combination with GCV, the recombinant adenovirus produces significant killing effect against SMMC-7721 cells in vitro.

Adenoviridae ; genetics ; Cell Line, Tumor ; Genetic Therapy ; Genetic Vectors ; Humans ; Liver Neoplasms ; therapy ; Thymidine Kinase ; genetics

BACKGROUNDSuicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses.

METHODSpAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E. coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease PacI and polymerase chain reaction (PCR), plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with PacI and sequently transfected into human embryo kidney 293 cells (HEK293) using Lipofectamine 2000.

RESULTSComet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture. After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk.

CONCLUSIONThe replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.

Adenoviridae ; genetics ; Bacteria ; genetics ; Defective Viruses ; genetics ; Genetic Therapy ; methods ; Green Fluorescent Proteins ; genetics ; Recombination, Genetic ; Thymidine Kinase ; genetics ; Virus Replication

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

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

Methanol fuel is a most promising substitute for gasoline. It is scarcely reported about methanol-fueled exhaust on the health effect, neither about genotoxicity research between methanol- and gasoline-fueled exhaust. In the present study, the two kinds of exhaust were sampled directly from tailpipe at the same type bus, the same state, L5178Y thymidine kinase (TK) gene mutation assay was used to investigate their genotoxicity at the same dose range, and compared with micronucleus and comet assay. The results showed that the genotoxicity of gasoline-fueled exhaust is stronger than that of methanol-fueled exhaust, while the cytotoxicity of methanol-fueled exhaust is stronger than that of gasoline-fueled exhaust at dose range. The study demonstrated that L5178Y TK gene mutation assay is more sensitive than micronucleus and comet assay.
Gasoline ; adverse effects ; Humans ; Methanol ; adverse effects ; Motor Vehicles ; Mutagenicity Tests ; Mutation ; Thymidine Kinase ; genetics ; Vehicle Emissions ; adverse effects

OBJECTIVE: To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1). METHODS: Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC₅₀) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay. RESULTS: The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group (P<0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC₅₀ of CA46-C23-KD cells to adriamycin was (0.147±0.02) μg/ml, which was significantly lower than (0.301±0.04) μg/ml of CA46-C23-KNC cells and (0.338±0.05) μg/ml of CA46 cells (P<0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G₂/M phase also decreased, while G0/G1 phase increased in cell cycle. CONCLUSION The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.
Humans ; Leukocytes, Mononuclear/metabolism* ; Apoptosis ; Cell Line, Tumor ; Lymphoma ; Thymidine Kinase/pharmacology* ; Doxorubicin/pharmacology* ; Cell Division ; RNA, Messenger/genetics*

OBJECTIVETo investigate the targeted killing effect of human telomerase reverse transcriptase promoter (hTERTp)/tk gene on human nasopharyngeal carcinoma (NPC) cells.

METHODSThe recombinant plasmid hTERTp/tk/pGL3 was transfected into human NPC HNE1 cells and the expressions of TK and telomerase were investigated. The targeted killing effect induced by hTERTp/tk on HNE1 cells was assessed using RT-PCR and MTT assay.

RESULTSTK gene expression was detected in HNE1 cells transfected by hTERTp/tk/pGL3, and the cells showed reduced telomerase and hTERT expression as compared with the control cells. hTERTp/tk/pGL3 resulted in target killing of HNE1 cells but not of the normal control cells. The tumor cell-killing effect of hTERTp/tk/pGL3 was slightly milder than that of the positive control CMV/tk/pGL3 that produced nonselective cell killing.

CONCLUSIONhTERTp/tk, a tumor-specific expression system, allows targeted tumor cell killing and reduces the activity of telomerase in NPC cells in vitro.

Cell Line, Tumor ; Gene Targeting ; Genetic Therapy ; methods ; Humans ; Nasopharyngeal Neoplasms ; enzymology ; genetics ; pathology ; Promoter Regions, Genetic ; genetics ; Telomerase ; genetics ; Thymidine Kinase ; genetics ; metabolism ; Transfection

To establish lentivirus-mediated system of double suicide genes and explore its killing effects on K562 cells, lentivirus transfer vector for double suicide genes was constructed using molecular methods, three plasmids of lentivirus gene transfer vector system were transferred into packaging cell line 293T using lipofectine method, the transfer effect was observed through fluorescence microscopy, the lentivirus particles were observed by means of electron microscopy. High titer of lentivirus was harvested from the supernatant of virus-producing cell culture and concentrated by high-speed centrifugation with Poly-L-Lysine (PLL). The K562 cells were infected with the concentrated supernatant containing the virus with the double suicide genes. Fluorescence microscopy and RT- PCR confirmed the integration and expression of extraneous gene. The cytotoxicity to these transgenic cells treated with 5-FC and GCV was measured by MTT assays. The growth inhibition ratio (GIR) of cells and inhibition concentration 50 (IC(50)) were counted. After administration of GCV and 5-FC, the changes of those cells were observed through scanning electron microscope. The results showed that lentivirus transfer vector with double suicide genes was constructed successfully. The above-mentioned plasmids were effectively transferred into 293T cells. So much green fluorescence was observed through fluorescence microscope. A lot of lentivirus particles were observed through transmission electron microscope. Double suicide genes mediated by lentivirus were stably integrated and expressed in K562 cells after infection with the concentrated virus using fluorescence microscopy and RT-PCR. The GIR of K562 cells using GCV or 5-FC was 48.73% or 50.69% respectively and it was apparently higher than that of untransfected cells (P < 0.01). When using GCV and 5-FC together, the GIR was 87.69%, which was apparently higher than that of group using GCV or 5-FC alone (P < 0.01). In conclusion, lentivirus-mediated gene transfer system could transfer CD and TK double suicide genes into K562 cells with high efficiency and it had strong killing effects when giving 5-FC and/or GCV. The cytotoxic effects of double suicide genes were superior to that of single suicide gene. The lentivirus-mediated double suicide gene transfer system is a high-efficiency gene transfer vector.
Cytosine Deaminase ; genetics ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genetic Therapy ; Genetic Vectors ; genetics ; HIV-1 ; genetics ; Humans ; K562 Cells ; Thymidine Kinase ; 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

To construct a TK-/gG- mutant of pseudorabies virus, the gG-detected transfer vector pUSKKBB and genomic DNA of pseudorabies virus TK-/gG-/LacZ+ were co-transfected into IBRS-2 cells. Transfection progeny were plated onto PK-15 cells and incubated for 2 days under methylcellulose. Then the overlay was removed and replaced by 1% low melting point agarose in DMEM supplemented with 150 microg/mL X-gal. After 2 days, white plaques were screened for and purified 4 times. By PCR amplification of gG-deleted gene and LacZ gene, a recombinant virus with TK-/gG- phenotype was confirmed. Sequence of the PCR product revealed that there were 1,176 bp detection in gG gene of the PRV TK-/gG- mutant. Amplifying the gG-deleted gene of different generations of the TK-/gG- mutant showed that the mutant was stable within PK-15 cells. TCID50 assay indicated that the recombinant virus grows well on PK-15 cells. The mice immunized with the TK-/gG- virus showed no sign of abnormality. As a control, all mice inoculated with PRV strain died from the infection. All mice that received TK-/gG- survived after a lethal PRV challenge. However none of the mice injected with phosphate-buffer saline (PBS) survived from the challenge. The above results demonstrated that the recombinant virus could be a candidate marker vaccine strain for eradicating pseudorabies in pig herds.
Animals ; Herpesvirus 1, Suid ; genetics ; pathogenicity ; Mice ; Mice, Inbred BALB C ; Mutation ; Pseudorabies Vaccines ; immunology ; Swine ; Thymidine Kinase ; genetics ; Vaccines, Synthetic ; Viral Envelope Proteins ; genetics ; immunology