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*

Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.
Antiviral Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cells, Cultured ; Dependovirus ; genetics ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Ganciclovir ; pharmacology ; Gene Expression Regulation, Neoplastic ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; genetics ; Humans ; In Situ Nick-End Labeling ; Luciferases ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics ; Pulmonary Alveoli ; cytology ; metabolism ; Pulmonary Surfactant-Associated Protein A ; genetics ; metabolism ; Thymidine Kinase ; genetics ; metabolism

OBJECTIVETo study the effection of suppression murine melanoma growth by Intratumor injection of recombinant attenuated salmonella carrying heat shock protein 70 and herpes simplex virus thymidine kinase genes.

METHODSPlasmids PCMV-mtHSP70-IRES-TK were electro-transferred into salmonella typhimurium SL7207 to construct recombinant salmonella typhimurium. In vivo, Recombinant bacteria were injected into the mouse melanoma and the antitumor effection was observed. The survival period was recorded and safety analysis for this vaccine in each group.

RESULTSIn vivo, the mtHSP70/HSV-tk recombinant bacteria can suppress tumor growth significantly and extend survival. After recombinant Salmonella, 10(9) CFU/mL, was administered as an intratumoral injection, No diarrhea were observed. During therapy, body weight did not change markedly.

CONCLUSIONResults of the animal experiment suggests intratumor injection of recombinant attenuated salmonella typhimurium containing mtHSP70 and HSV-tk genes, has targeting ability against B16 tumor cell and could significantly inhibit tumor growth .

Animals ; Bacterial Proteins ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; pharmacology ; Genetic Therapy ; methods ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Melanoma, Experimental ; microbiology ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Mycobacterium tuberculosis ; genetics ; Salmonella typhimurium ; genetics ; immunology ; Simplexvirus ; enzymology ; genetics ; Skin Neoplasms ; therapy ; Thymidine Kinase ; genetics ; immunology ; Vaccines, Attenuated ; genetics ; immunology ; pharmacology ; Vaccines, DNA ; genetics ; immunology ; pharmacology

OBJECTIVETo observe the gene expression of herpes simplex virus type 1 thymidine kinase (HSVl-tk) in rat malignant ovarian tumor tissues and the therapeutic effect of ganciclovior (GCV) after intra-arterial infusion of HSVl-tk gene therapy mediated by GE7-delivery system.

METHODSA GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 4-element complex was constructed. Eighteen rats with DMBA-induced ovarian tumor were divided into 3 groups as Atk, ANS and Vtk groups. The 4-element complex GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 was injected via the ovarian artery into the rats of Atk group, saline buffer was injected in the ANS groups, and the 4-element complex was injected via the tail vein into the rats of Vtk group. All rats received intraperitoneal injection of GCV in a dose of 50 mg/kg daily for 10 days. The rats were sacrificed 3 days after the final dose of GCV, and the tumor weight was measured and tumor growth inhibition rate was calculated. Flow cytometry was used to assess the cell cycle and apoptosis.

RESULTSThe tumor weight in the rats of Atk group was (4.77 ± 2.31) g, significantly lower than that of ANS group [(14.66 ± 6.26) g, P < 0.01] and Vtk group [(17.53 ± 7.19) g, P < 0.01]. The tumor growth inhibition rate of the Atk group was 67.5%, while that of Vtk group was -19.6%. The flow cytometry showed that S-phase tumor cells in the Atk group were (54.32 ± 9.65)%, significantly higher than that in the ANS (27.43 ± 9.22)% and (30.16 ± 11.57)% in the Vtk group (both P < 0.01). The tumor cell apoptosis rate in the Atk group was (39.15 ± 12.16)%, significantly higher than that in the ANS group [(11.86 ± 5.28)%, P < 0.01] and Vtk group [(14.32 ± 6.43)%, P < 0.01].

CONCLUSIONHSV1-tk/GCV gene therapy system mediated by GE7 non-viral delivery system via ovarian arterial infusion effectively causes cell cycle arrest at S phase and enhances cell apoptosis, therefore, exerts an inhibitory effect on tumor growth.

9,10-Dimethyl-1,2-benzanthracene ; Adenocarcinoma ; chemically induced ; pathology ; therapy ; Animals ; Antiviral Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Female ; Ganciclovir ; pharmacology ; Gene Transfer Techniques ; Genetic Therapy ; Herpesvirus 1, Human ; genetics ; metabolism ; Infusions, Intra-Arterial ; Ovarian Neoplasms ; chemically induced ; pathology ; therapy ; Random Allocation ; Rats ; Rats, Wistar ; Thymidine Kinase ; genetics ; metabolism

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

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

OBJECTIVE: To investigate the mechanism and effect of diallyl disulfide (DADS) on the bystander effect of herpes simplex virus kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy system which was mediated by recombinant adeno-associated virus (rAAV) in lens epithelial cells. METHODS: Immunohistochemistry method was used to determine the distribution of connexin 43 (Cx43) protein in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene. Cx43 protein was measured and analyzed in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene that was induced by various DADS. Cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: DADS increased the Cx43 protein expression in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene, and especially in 50 μmol/L DADS. After combining with DADS, the bystander effect was significantly improved (P<0.05). CONCLUSION DADS can elevate the expression of Cx43 protein and enhance the bystander effect of HSV-tk/GCV suicide gene therapy system.
Adenoviridae ; genetics ; metabolism ; Allyl Compounds ; pharmacology ; Animals ; Antiviral Agents ; pharmacology ; Bystander Effect ; drug effects ; Cells, Cultured ; Connexin 43 ; metabolism ; Disulfides ; pharmacology ; Epithelial Cells ; metabolism ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Lens, Crystalline ; cytology ; drug effects ; metabolism ; Plant Oils ; Rabbits ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism

OBJECTIVETo study the selective cytotoxic effect of lentivirus-mediated double suicide gene (CD/TK) against human gastric carcinoma cells SGC-7901 in vitro.

METHODSSGC-7901 cells were infected with FGW-KDRP-CD/TK vector and the infection efficiency was observed under a fluorescence microscope. The morphological changes of the infected cells were observed by Giemsa staining. Flow cytometry (FCM) was employed for cell cycle analysis, and the expression of CD/TK was detected by RT-PCR. The infected cells were then treated with the prodrugs ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT method.

RESULTSThe infection efficiency of the lentiviral vector in SGC-7901 cells increased with the titer of the virus, which produced no significant effect on the cancer cell morphology in vitro or on the percentages of G0-G1, G2-M and S phase cells (P>0.05). RT-PCR demonstrated the expression of CD/TK gene in SGC-7901 cells infected by FGW-KDRP-CD/TK. The infected cells were highly sensitive to the prodrugs with a dose-dependent cytotoxic effect within a specific concentration range of the drugs, whereas the non-infected cells were not sensitive to the prodrugs. Combined use of the two prodrugs produced an obviously stronger inhibitory effect than either of the them (P<0.05). When combined, GCV and 5-FC at the concentration of 0.1+40, 1+80, 10+160, and 100+320 mg/L demonstrated a synergetic effect with a CDI<1.

CONCLUSIONLentivirus-mediated CD/TK fusion gene system can selectively kill gastric cancer cells, and the two prodrugs show a synergistic cytotoxic effect.

Adenocarcinoma ; genetics ; pathology ; Cell Line, Tumor ; Cytosine Deaminase ; biosynthesis ; genetics ; Cytotoxins ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Stomach Neoplasms ; genetics ; pathology ; Thymidine Kinase ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

OBJECTIVETo evaluate the effect of HSV-tk gene transfer on the apoptosis of fibroblast in rats with scald injury.

METHODSThe recombinant eukaryotic expression vector pcDNA3.1/tk was transfected via liposome in the skin of rats with scald injury. The expression of tk gene was detected by RT-PCR technique, and after GCV injection, the apoptosis of the fibroblasts positive for tk gene was observed under transmission electron microscope.

RESULTSLiposome-mediated HSV-tk gene transfection of the rat skin resulted in the positive expression of tk gene in the fibroblasts in the burn wound. GCV injection induced the apoptosis of the positively transfected fibroblasts.

CONCLUSIONHSV-tk gene transfer mediated by liposome can promote the apoptosis of the fibroblasts in rats with scald injury.

Animals ; Apoptosis ; drug effects ; Burns ; pathology ; Fibroblasts ; pathology ; Gene Transfer Techniques ; Liposomes ; pharmacology ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Simplexvirus ; genetics ; Thymidine Kinase ; genetics

OBJECTIVETo investigate the potentiality of herpes simplex virus thymidine kinase transduced endothelial progenitor cells (EPC-TK) as angiogenesis-targeting vector in the glioma treatment in vitro and in vivo.

METHODSEPC-TK were mixed with human umbilical vein endothelial cells (HUVECs), U87 or U251 cells at various ratios for ganciclovir (GCV) treatment. The bystander effect was observed by counting the survival cells using MTT assay, and the apoptotic cells were determined by annexin-V and propidium iodide (PI) staining. EPC-TK, EPCs, or phosphate buffered saline (PBS) were injected into the nude mice model of glioma xenograft by tail vein, for the EPC-TK group, EPC group, and PBS group, respectively. And then the changes of tumor volume and tumor vasculature were observed.

RESULTSGCV killed most EPC-TK and reduced the number of other viable cells in a cell:cell ratio-dependent and time-dependent manner. EPC-TK obviously inhibited tumor growth. The tumor volumes on day 21 were 1741.20+/- 576.10 mm(3), 3275.52 +/- 710.86 mm(3) and 3033.09+/-1134.86 mm(3) in the EPC-TK, EPC and PBS group, respectively. EPC-TK also displayed a significant effect on the inhibition of tumor angiogenesis.

CONCLUSIONEPC-TK can exert a potent antiglioma effect via inhibiting angiogenesis.

Angiogenesis Inhibitors ; pharmacology ; Animals ; Antiviral Agents ; pharmacology ; Bystander Effect ; Cell Transformation, Viral ; physiology ; Endothelial Cells ; virology ; Endothelium ; Genetic Vectors ; Glioma ; therapy ; Humans ; Mice ; Mice, Nude ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; genetics ; Transduction, Genetic ; Transfection ; Xenograft Model Antitumor Assays