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

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

OBJECTIVEAdenovirus vector-mediated herpes simplex virus thymidine kinase gene (ADV-TK) transfer in combination with ganiciclovir (GCV) is one of the major gene therapy strategies to eradicate tumor cells. This study was aimed at determining the in vivo anti-tumor efficacy of ADV-TK in combination with ganiciclovir (GCV).

METHODSA murine xenotransplant model of human small-cell lung cancer was established. ADV-TK was administrated by intra-tumoral injection followed by intraperitoneal administration of GCV. The anti-tumor efficacy was evaluated using index of tumor volume, relative tumor volume, tumor weight, relative tumor proliferative rate, and tumor growth curve.

RESULTSIn the presence of GCV, ADV-TK effectively inhibited growth of human small-cell lung cancer in a dose-dependent fashion. An inhibition plateau was not observed within the current dosage range. ADV-TK at a dose of 6.0 x 10(9) viral particles/kg in the presence of GCV lead to 64.6% and 81.7% inhibition of tumor growth respectively in two independent experiments. ADV-TK or GCV alone caused slight inhibition of tumor growth, which was not statistically significant as compared to the negative control group (P > 0.05).

CONCLUSIONADV-TK followed by GCV is highly efficacious to inhibit the growth of human small-cell lung cancer in a murine xenotransplant model. The results presented here are encouraging to warrant a further clinical evaluation of the potential therapeutic benefits of this strategy.

Adenoviridae ; genetics ; Animals ; Carcinoma, Small Cell ; therapy ; Female ; Ganciclovir ; therapeutic use ; Genetic Therapy ; Humans ; Lung Neoplasms ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; Transplantation, Heterologous

The potential therapeutic benefit of introducing IFN-gamma and GM-CSF genes in combination with the HSVtk suicide gene into subcutaneously implanted CT26 tumor cells was compared with that from each treatment alone. Cells, unmodified or retrovirally transduced with HSVtk or IFN-gamma/GM-CSF genes, were inoculated subcutaneously into syngeneic BALB/c mice in various combinations. HSVtk gene, with intraperitoneal ganciclovir treatment, reduced tumor volume by 81% at locally inoculated tumor sites (p<0.01) and by 25% at distantly inoculated tumor sites (p=0.052). IFN-gamma/GM-CSF genes showed a 56% tumor volume reduction at local tumor sites (p<0.01) and 15% volume reduction at remote tumor sites, although this was not statistically significant. The combination of HSVtk (with GCV) and IFN-gamma/GM-CSF genes showed an 81% volume reduction at local tumor sites (p<0.01) and a 43% volume reduction at remote tumor sites (p<0.01). Thus, the combination of HSVtk and IFN-gamma/GM-CSF gene therapy produced greater therapeutic efficacy than either treatment alone.
Animals ; Cell Line ; Cell Line, Tumor ; Gene Therapy/*methods ; Genes, Transgenic, Suicide ; Granulocyte Macrophage Colony-Stimulating Factors, Recombinant/biosynthesis/*genetics/therapeutic use ; H-2 Antigens/metabolism ; Interferon-gamma, Recombinant/biosynthesis/*genetics/therapeutic use ; Male ; Mice ; Mice, Inbred BALB C ; Neoplasms, Experimental/*therapy ; Research Support, Non-U.S. Gov't ; Simplexvirus/enzymology/genetics ; Thymidine Kinase/*genetics/therapeutic use ; Transduction, Genetic

OBJECTIVETo evaluate the feasibility and efficacy of cytocine deaminase-thymidine kinase (CD-TK) fusion double suicide gene therapy using adenovirus mediated CD-TK gene and green fluorescent rotein (GFP) gene combined with ganciclovir(GCV) or 5-flourocytosine(5-FC) in a murine subcutaneous bladder carcinoma model.

METHODSA replication defective adenovirus vector containing CD-TK gene was used. Subcutaneous tumors were established in syngenic C57BL/6 female mice with 1 x 10(6) Mb49 cells. Intratumoral injection of AdCD-TK (1.58 x 10(8) PFU, qd x days) in combination with GCV (40 mg.kg(-1).d(-1), ip, qd x 10 days) or 5-FC (400 mg.kg(-1).d(-1), ip, qd x 10 days) was administered in vivo for the determination of treatment efficacy in separate controlled experiments.

RESULTSIn vivo experiments demonstrated that the mean volume of tumor in the group of AdCD-TK/GCV(326.58+/-109.56 mm(3)), AdCD-TK/5-FC (235.33+/-62.94 mm(3)) and AdCD-TK/(GCV+5-FC) (23.58+/-6.78 mm(3)) was reduced significantly compared with that of control group (993.51+/-158.32 mm(3)) (P=0.00), the mean volume of tumor in the group of AdCD-TK/(GCV+5-FC) was significantly less than that in the group of AdCD-TK/GCV or AdCD-TK/5-FC (P=0.04). Tumor necrosis was revealed by histomorphology compared with control animals.

CONCLUSIONSAdenovirus mediated CD-TK double suicide gene combining with GCV or 5-FC could provide an effective therapy in an experimental murine bladder carcinoma by significantly inhibiting tumor growth. The treatment efficacy of AdCD-TK combining GCV and 5-FC was superior to that of AdCD-TK combining GCV or AdCD-TK combining 5-FC.

Adenoviridae ; genetics ; Animals ; Cell Line ; Cell Line, Tumor ; Cytosine Deaminase ; genetics ; metabolism ; Defective Viruses ; genetics ; Female ; Flucytosine ; pharmacology ; therapeutic use ; Ganciclovir ; pharmacology ; therapeutic use ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Thymidine Kinase ; genetics ; metabolism ; Treatment Outcome ; Urinary Bladder Neoplasms ; pathology ; therapy

OBJECTIVETo evaluate the effect of Avastin and adenovirus-thymidine kinase/ Ganciclovir (Ad-TK/GCV) suicide gene system on nasopharyngeal carcinoma in vivo and in vitro.

METHODSThe expression of vascular endothelial growth factor (VEGF) by CNE1 cell line was detected by VEGF ELISA. The effect of Avastin and Ad-TK/GCV on CNE1 cell was detected by methyl thiazolyl tetrazolium (MTT) assay. Flow cytometry analysis and Hoechst 33342 staining were adopted to explore the killing mechanism of Ad-TK/GCV. The nude mice models of CNE1 cell xenografts were established. After intra-tumoral injection of PBS, Avastin, Ad-TK/GCV, Ad-Lac-Z/GCV and Ad-TK/GCV + Avastin, tumor volume was measured and tumor inhibitory rate was calculated. Then the tumors were removed and subjected to histological examination.

RESULTSCNE1 cells could produce VEGF. Avastin had no direct effect on CNE1 cells. The killing effect of Ad-TK/GCV increased with the increase of Ad-TK multiple of infection and the prodrug concentration, which was enhanced by the existence of bystander effect. Compared with control group, the death cell rate (P = 0.000) and apoptosis cell rate (P = 0.000) had significant difference. The study in vivo showed the tumors treated with Avastin, Ad-TK/GCV and Ad-TK/GCV + Avastin grew slowly compared with control. Tumor volume of treated groups was significantly smaller than that of control (all P < 0.05 or P = 0.000). Tumor weight of treated groups was significantly lower than that of control (all P = 0.000). The histological examination showed local necrosis in Ad-TK/GCV group and Ad-TK/GCV + Avastin group, poor angiogenesis in Avastin group and Ad-TK/GCV + Avastin group.

CONCLUSIONSAvastin had no direct effect on CNE1 cells in vitro. Ad-TK/GCV suicide gene system killed NPC cells by inducing cell necrosis and apoptosis, which could be enhanced by the existence of bystander effect. Avastin and Ad-TK/GCV suicide gene system could inhibit the growth of NPC CNE1 cell xenografts. Combination therapy had a synergic effect.

Adenoviridae ; enzymology ; genetics ; Animals ; Antibodies, Monoclonal ; therapeutic use ; Antibodies, Monoclonal, Humanized ; Bevacizumab ; Female ; Ganciclovir ; Genes, Transgenic, Suicide ; Genetic Therapy ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nasopharyngeal Neoplasms ; therapy ; Thymidine Kinase ; genetics ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; genetics ; therapeutic use

To investigate the antitumor effect of herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system on human bladder cancer cells (T24), a retroviral vector with the gene (pLXSN-TK) was transduced into the packaging cell line PA317. A nude mouse model with human T24 was established to examine the in vivo efficacy. The animals were randomly assigned to two treatment groups and two control groups. Treatment I and Treatment II were given in situ injection of virus suspension and PA317/TK respectively, followed by treatment with GCV for 14 days. Control I and Control II were given in situ injection of same volume of normal saline and PA317/TK respectively, followed by treatment with GCV and with normaly physiologic saline respectively for 14 days. The weight and the volume of tumor were measured. HSV-TK mRNA expression was determined by hybridization in situ. Cell apoptosis was evaluated by flow cytometry (FCM) and termininal deoxynucleofidyl transferase-mediated dUTP nick end labelling (TUNEL) technique. The results showed: (1) In vivo, the retrovirus transferred HSV-TK gene can be transduced into human bladder cancer cell T24. The tumors in T24 mice with TK gene transduced were much smaller than those in other groups. (2) After treatment with HSV-TK/GCV, the phenomenon of bladder ceancer cell apoptosis was more conspicuous as compared with that of other groups. Therefore, HSV-TK/GCV system can suppress the growth of T24 in vivo and may relate to "bystander effect". It could be a valuable therapy for human bladder cancer.
Animals ; Antiviral Agents ; therapeutic use ; Ganciclovir ; therapeutic use ; Gene Transfer Techniques ; Genetic Therapy ; Herpesvirus 1, Human ; enzymology ; genetics ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Random Allocation ; Thymidine Kinase ; genetics ; Tumor Cells, Cultured ; Urinary Bladder Neoplasms ; genetics ; pathology ; therapy

OBJECTIVETo study target killing of 5-FU drug-fast cancer cells with thymidylate synthase (TS) and p16 gene promoters inducting TK gene expression.

METHODSTS promoter was inserted to 5' end and p16 promoter inserted to 3' end of TK cDNA sequence, constructing recombinant plasmid of pXJ41. Human rectal cancer cell lines of HR-8348 and normal peripheral blood mononuclear cells (PBMC) were transfected with the recombinant plasmid. Plating efficiency was counted and survival rates of cells were tested with MTT method. And suppression rates of xenograft tumors in nude mice were examined.

RESULTSRecombinant pXJ41 with double promotors and TK gene was transfected into HR-8348, and positive expression of TS and TK was observed. The expression of TK gene was consistent with TS expression. Plating efficiency was 9/300, 92/300 in transfected HR-8348 and contrast cells respectively (t = 33.885, P < 0.01). Cancer cell growth rate reduced markedly in the transfected group. The suppression rate of xenograft tumor growth was 74.5%. With the recombinant pXJ41 to transfect PBMC, p16 expression was positive, but TK and TS expressions were negative. No damnification was observed in PBMC.

CONCLUSIONSTS and p16 double promoters are capable of inducting TK target killing of 5-FU drug-fast cancer cells, thus protecting normal cells and improving safety of gene therapy.

Animals ; Antimetabolites, Antineoplastic ; metabolism ; pharmacology ; therapeutic use ; Disease Models, Animal ; Drug Delivery Systems ; Fluorouracil ; metabolism ; pharmacology ; therapeutic use ; Gene Transfer Techniques ; Genes, p16 ; Genetic Therapy ; Humans ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neoplasms, Experimental ; drug therapy ; Promoter Regions, Genetic ; Thymidine Kinase ; genetics ; Thymidylate Synthase ; genetics ; metabolism ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays