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 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

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

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

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 investigate in vitro and in vivo bystander effect, including distance bystander effect of adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene (HSV-tk).

METHODSIn vitro, mixed tk + BEL-7402 cells and tk-BEL-7402 cells in diverse proportions and ganciclovir (GCV) was given, then tested the survival ratio of cells by MTT. In vivo, 5 x 10(6) and 5 x 10(7) tk + BEL-7402 cells were injected into the tumors in nude mice following GCV. The change of the size of the tumors was observed. To observe distance bystander effect, adenovirues with HSV-tk (1 x 10(9) PFU) were injected into the tumor, which was the one of the bilateral tumors in nude mice. Subsequently, GCV was given and the change of the tumor was observed.

RESULTSSignificant bystander effect was observed in vitro and in vivo. In vitro when tk + cells: tk(-) cells was 1:9, the survival rate of mixed cells was 36.6%. When the proportion of tk(+) cells was 90%, the survival rate of mixed cells was 3.2%. In vivo, those tumors with injection of tk(+) BEL-7402 cells were suppressed (P < 0.05). But in group of distance bystander effect the tumors on another side were not suppressed.

CONCLUSIONIn vitro, bystander effect exists. In nude mice, if tk(+) cells and tk(-) cells are contiguous, bystander effect is significant, or probably no bystander effect.

Adenoviridae ; genetics ; Animals ; Bystander Effect ; Disease Models, Animal ; Ganciclovir ; therapeutic use ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neoplasms, Experimental ; drug therapy ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Tumor Cells, Cultured

OBJECTIVETo investigate enhancement of the bystander effect by tanshinone IIA (Tan) in HSV-tK/GCV system and the correlation with expression of connexin 43 mRNA.

METHODSThe cytotoxic effect in HSV-tK/GCV in cervical carcinoma cell line ME180 (ME) and ME/TK was examined by MTT assays. Cx43 mRNA expression was detected by fluor-quantitative RT-PCR.

RESULTSTan markedly increased sensitivity of ME/TK cells for GCV in HSV-tK/GCV system. In the presence of 2 micro g/ml GCV, compared with the absence of Tan (0 mol/L), an obvious decrease in survival rate was seen at any given mixture of ME and ME/TK cells exposed to 1.3 x 10(-9) mol/L Tan. Statistics showed significant difference (P < 0.05). However, enhancement of bystander mediated cell killing occurred only in the range of Tan concentrations used (1.3 x 10(-8), 1.3 x 10(-9) mol/L). RT-PCR showed that the ratio of relative copy number of Cx43 mRNA increased by 8.83 and 8.47-fold in ME cells exposed to 1.3 x 10(-8) and 1.3 x 10(-9) mol/L Tan, respectively.

CONCLUSIONFor the first time we report that in cervical carcinoma ME180 cell line, Tan possesses a remarkable enhancing role on the bystander effect in the HSV-tK/GCV system. It is associated with up-regulation of Cx43 mRNA expression.

Antineoplastic Agents, Phytogenic ; pharmacology ; Bystander Effect ; Cell Line, Tumor ; Cell Survival ; drug effects ; Connexin 43 ; genetics ; Diterpenes, Abietane ; Female ; Ganciclovir ; pharmacology ; Genetic Therapy ; Humans ; Phenanthrenes ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; Uterine Cervical Neoplasms ; therapy