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

OBJECTIVETo study the effect of cotransfection of genes of insulin-like growth factor I (IGF-I) and herpes simplex virus thymidine kinase (HSV-tk) on wound healing.

METHODSThirty male Wistar rats were inflicted with 30% TBSA full-thickness scald. They were then divided into A group (4.6 microg pcDNA3.1/IGF-I+Lipofectamine 2000+saline), B group (3.6 microg pcDNA3.1/HSV-tk+Lipofectamine 2000+saline), C1 group and C2 group (2.3 microg pcDNA3.1/IGF-I+1.8 microg pcDNA3.1/HSV-tk+Lipofectamine 2000+saline), and D group (3.0 microg pcDNA3.1+Lipofectamine 2000+saline) according to the random number table, with 6 rats in each group. The above-mentioned mixtures were subcutaneously injected into left back of each rat the moment after injury and on post scald day (PSD) 7, 14, 21, and 28. Gancyclovir (2.5 mg/100 g) was hypodermically injected into rats in C2 group on PSD 29, 30, 31, 32. Changes in body weight of rats were measured. Wound healing rates were calculated. On PSD 35, the expressions of IGF-I gene in local wound and liver tissue were determined with immunohistochemical staining. The serum expression of IGF-I was determined with radioimmunoassay. Expression of HSV-tk gene in local wound was determined with RT-PCR. Apoptosis of fibroblast in C1 and C2 groups was observed under transmission electron microscope. Data were processed with one-way analysis of variance and Turkey method.

RESULTSBody weight of rats in A, C1, and C2 groups increased from PSD 7 through 35, and the difference between former three groups and B, D groups was statistically significant (with F value respectively 2.764, 4.519, 5.009, 13.449, 5.877, P values all below 0.05). Wound healing rates of rats in A, C1, and C2 groups were higher than those in B, D groups (with F value respectively 5.286, 100.880, 152.380, 127.850, 147.750, P values all below 0.05). IGF-I gene was positively expressed in wound fibroblast in A, C1 and C2 groups, but negatively in liver tissues of all the rats. There was no significant statistical difference among groups in serum content of IGF-I [from (1185+/-170) to (1270+/-130) ng/mL, F=0.355, P=0.838]. HSV-tk gene was positively expressed in rat skin tissue in B, C1 and C2 groups. Fibroblast apoptosis was observed under transmission electron microscope in C2 group, but it was not observed in C1 group.

CONCLUSIONSCotransfection of pcDNA3.1/IGF-I and pcDNA3.1/HSV-tk mediated by liposome can promote wound healing, and inhibit the scar proliferation to some extent.

Animals ; Burns ; genetics ; metabolism ; therapy ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Male ; Rats ; Rats, Wistar ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Transfection ; Wound Healing

OBJECTIVETo establish a highly sensitive and specific dual-color fluorescence in situ hybridization (D-FISH) method used for chromosomal localization of foreign genes in double transgenic mice.

METHODSTwo strains of double transgenic mice were used in this experiment, one was integrated with the herpes simplex virus thymidine kinase (HSV-tk) and the enhanced green fluorescence protein (eGFP), the other was with the short hairpin RNA interference(RNAi) and beta(654). Splenic cells cultured in vitro were arrested in metaphase by colchicine and hybridized with digoxigenin-labeled and biotinylated DNA probes, then detected by rhodamine-conjugated avidin and FITC-conjugated anti-digoxigenin.

RESULTSDual-color fluorescence signals were detected on the same metaphase in both transgenic mice strains. In HSV-tk/eGFP double transgenic mice, strong green fluorescence for HSV-tk and red for eGFP were observed and localized at 2E5-G3 and 8A2-A4 respectively. In beta(654)/RNAi mice, beta(654) was detected as red fluorescence on chromosome 7D3-E2, and RNAi showed random integration on chromosomes. It was detected as green fluorescence on chromosome 12B1 in one mouse, while on 1E2.3-1F and 3A3 in the other.

CONCLUSIONHighly sensitive and specific D-FISH method was established using the self-prepared DNA probes, and chromosomal localization of the foreign genes was also performed in combination with G-banding in double transgenic mice. This technology will facilitate the researches in transgenic animals and gene therapy models.

Animals ; Cells, Cultured ; Color ; Green Fluorescent Proteins ; genetics ; In Situ Hybridization, Fluorescence ; methods ; Mice ; Mice, Transgenic ; Physical Chromosome Mapping ; methods ; Sensitivity and Specificity ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; Transgenes

OBJECTIVETo investigate the therapeutic effects of adenovirus-mediated transfer of the herpes simplex virus thymidine rinase gene (HSV-tk) used by several methods and the dose-effect relationship.

METHODSDiverse doses (1 x 10(9) PFU, 1 x 10(10) PFU, 1 x 10(11) PFU) of adenovirus-mediated transfer of HSV-tk were given by intraparenchymatous, intravenous and intraperitoneal injection, and ganciclovir (GCV) (100 mg.kg(-1).d(-1)) was injected into the cavity of the peritoneum to treat human hepatocarcinoma. The change of tumors size was observed and the fragments of HSV-tk gene were tested.

RESULTSIn nude mice after intraparenchymatous injection and high-dose (1 x 10(11) PFU) intravenous injection, the tumors were suppressed significantly (t = 13.1, 12.4, P < 0.01) and lots of fragments of HSV-tk gene were observed. In mice after intraperitoneal injection and low-dose (1 x 10(9) PFU, 1 x 10(10) PFU) intravenous injection, no suppressive effect was observed (t = 1.8, 1.0, 2.1, 1.1, 0.8, P > 0.05) with few or without fragments in the tumors.

CONCLUSIONSAdenovirus-mediated transfer of the HSV-tk by intraparenchymatous or intravenous injection is effective in treatment of hepatocarcinoma in nude mice, but intraperitoneal injection has no therapeutic effect.

Adenoviridae ; genetics ; Animals ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Humans ; Liver Neoplasms, Experimental ; therapy ; Mice ; Mice, Inbred BALB C ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; Tumor Cells, Cultured

Cytokines ; secretion ; Cytotoxicity, Immunologic ; Graft vs Leukemia Effect ; Humans ; Immunophenotyping ; Lymphocyte Activation ; Retroviridae ; genetics ; Simplexvirus ; enzymology ; T-Lymphocytes ; immunology ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVEEfficacy of HSV-tk/GCV system antitumor effects was assessed on human laryngeal cancer cell line Hep-2 in vitro. To assess the HSV-tk/CGV system whether has an antitumour effect on human laryngeal squamous cell cancer Hep-2 in vitro. The mechanisms of cytotoxity were also assessed.

METHODSHep-2 cells were transfected with HSV-tk gene by lipofection. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the HSV-tk gene expression. MTT was utilized to test for the cytotoxicity of this system. The cell-circle arrest and apoptosis were analyzed by flowcytometry assay.

RESULTSHSV-tk gene transfected cells demonstrated obvious cytoreductivity followed by ganciclovir (GCV) administration and this cytoreductivity showed partial GCV dose-independent. HSV-tk gene transfected cells demonstrated obvious s-phase arrest, no apoptosis and necrosis occurred.

CONCLUSIONSThe HSV-tk/GCV system can inhabit the growth of Hep-2 cells effectively. S-phase arrest perhaps is the main reason that leads to the cell inhibition in our study. HSV-tk/GCV system has potential antitumor effects for the future clinical practice.

Carcinoma, Squamous Cell ; therapy ; Cell Line, Tumor ; Ganciclovir ; Genes, Transgenic, Suicide ; Genetic Therapy ; Genetic Vectors ; Humans ; Laryngeal Neoplasms ; therapy ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVETo explore the HSVtk gene expression mediated by the retroviral vector and to obtain high titer recombinant retroviral virus.

METHODSThe recombinant vector pRevTRE/HSVtk was constructed by inserting HSVtk gene into pRevTRE. The recombinant retrovirus, which was produced from cloned PA317 cells screened by hygromycin B after "micro-pingpong" technique transferring with pRevTRE/HSVtk plasmids DNA by using modified calcium phosphate precipitation method. HSVtk gene expression was performed on target cells and virus titers were detected in different cultured temper, time and sodium butyrate concentration.

RESULTSThe recombinant retroviral vector pRevTRE/HSVtk was constructed and HSVtk gene expression was detected on target cells after they were infected with the recombinant retrovirus.

CONCLUSIONHigh titer of retroviruses could be obtained in the culture medium of PA317 cell line through "micro-pingpong" technique at 30 hours and 10 mmol/L sodium butyrate concentration followed by frozen ultrafiltration.

Animals ; Breast Neoplasms ; enzymology ; pathology ; virology ; Cell Line ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Enzymologic ; Genetic Vectors ; Humans ; Mice ; NIH 3T3 Cells ; Recombination, Genetic ; Retroviridae ; genetics ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; biosynthesis ; genetics ; Titrimetry ; Transfection

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

OBJECTIVETo observe the therapeutic effect of CDglyTK gene mediated by radiation-inducible promoters in the treatment of buccal carcinoma in Golden Hamster.

METHODSAnimal models of buccal carcinoma in golden hamster were established by painting 0.5% dimethyl-benzanthracene. The plasmids pcDNA (+) 3.1/E-CDglyTK were transfected into tumors by lipofectamine. 24 h later, the tumors were exposed to 3 Gy irradiation. Animals were monitored at regular intervals for volume of tumors. CDglyTK mRNA was assayed by RT-PCR. Apoptosis and proliferating cell nuclear antigen were detected respectively by in situ end-labeling and immunohistochemical methods.

RESULTSCompared with control groups, the tumor was suppressed obviously by CDglyTK gene therapy combined with 3 Gy induction radiation. The expression of CDglyTK gene could be detected by RT-PCR in the transfected tumor, and up-regulation of CDglyTK expression was found in tumor exposed to radiation (P < 0.05). There was significant difference in apoptosis index or proliferation index between tumor without irradiation and tumor with irradiation (P < 0.05).

CONCLUSIONSThe radiation-inducible promoter can be served as a molecular switch to regulate the expression of CDglyTK gene in buccal carcinoma in golden hamster, and low dose induction radiation can significantly improve the therapeutic effects.

Animals ; Carcinoma, Squamous Cell ; diagnostic imaging ; therapy ; Cheek ; diagnostic imaging ; Cricetinae ; Cytosine Deaminase ; genetics ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Mesocricetus ; Mouth Neoplasms ; radiotherapy ; therapy ; Promoter Regions, Genetic ; radiation effects ; Radiography ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics

OBJECTIVETo construct the localization system involving anti-TfR monoclonal antibody (McAb) and AFP promoters and assess its effect on human hepatoma cell lines.

METHODSThe conjugate of anti-TfR McAb and polylysine (PLL) was made by SPDP and purified by molecular screen chromatography. DNA blocking test determined that the ratio of one pEBAF/tk to six Ab-PLL was the most suitable to couple them. The pEBAF/tk recombinant plasmid bearing HSV-TK gene was coupled to Ab-PLL by noncovalent bond. The pEBAF/tk was transferred into human hepatoma cell line HepG2, SMMC7721 and pulmonary cancer cell line A549 by receptor-mediated gene delivery (Ab-PLL-DNA) and liposome procedure. The growth inhibitory rates of HepG2, SMMC7721 and A549 cells were measured by MTT assay.

RESULTSThe inhibitory rates of HepG2/tk in 100 mg/L and 1 mg/L of GCV were 60.5% and 24.3%, respectively. The inhibitory rate of GCV to SMMC7721 was 23.2% in 3 days. The pulmonary cancer cell A549, A549/tk (Ab) and A549 /tk (lipo) could not be inhibited by the addition of GCV.

CONCLUSIONThe localization system employed in this paper has high specificity, effectiveness and safety for gene therapy. It would be a promising strategy for gene therapy.

Antibodies, Monoclonal ; therapeutic use ; Carcinoma, Hepatocellular ; therapy ; Cell Line, Tumor ; Ganciclovir ; therapeutic use ; Genetic Therapy ; Humans ; Liver Neoplasms ; therapy ; Receptors, Transferrin ; immunology ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; alpha-Fetoproteins ; genetics