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

BACKGROUNDHerpes simplex virus thymidine kinase phosphorylates ganciclovir to ganciclovir monophosphate, which is then converted to ganciclovir triphosphate by endogenous cellular nucleoside kinases. The ganciclovir triphosphate acts as a DNA chain terminator due to the lack of a functional 3'-OH group and terminates the process of DNA replication, hence leading to cell apoptosis. At present, HSVtk gene usually acts as suicide gene to kill tumor cells. The aim of this study was to investigate the selective cytotoxicity of the herpes simplex virus thymidine kinase/ganciclovir (HSVtK/GCV) suicide gene system controlled by the a-fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) cells in vitro.

METHODSpAFP-HSVtk-IRES2-EGFP recombinant plasmid vectors driven by the AFP promoter were constructed. HL-7702 liver cells, HUH-7 HCC, and HepG2 HCC were transfected with the recombinant plasmids. HSVtK gene expression was detected using Western blotting analysis. HepG2 cells line stably expressing HSVtk gene was selected by G418 reagent. The cytotoxicity of HSVtK/GCV suicide gene system on hepatoma cells was measured by CCK-8 reagents when different doses of ganciclovir were added.

RESULTSPlasmid pAFP-TK-IRES2-EGFP-expressed HSVtk gene was constructed successfully. HSVtk gene expression level was significantly higher in AFP-positive hepatoma cells than in AFP-negative liver cells. After G418 selection, a HepG2 cells line stably expressing HSVtk gene was acquired. With the increase of the dose of ganciclovir the optical density at 450 nm of HepG2 cells stably expressing HSVtk gene gradually decreased (P < 0.05).

CONCLUSIONThe HSVtK gene-specific expression in hepatoma cells as well as the cytotoxicity of the suicide gene system in HepG2 cells provided the basis for the targeted gene therapy of HCC.

Apoptosis ; drug effects ; genetics ; Carcinoma, Hepatocellular ; genetics ; Cell Line, Tumor ; Fetal Proteins ; genetics ; Ganciclovir ; analogs & derivatives ; pharmacology ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; Promoter Regions, Genetic ; genetics ; Transfection

OBJECTIVETo monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods.

METHODSHCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing.

RESULTSHCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10(-4.618)/0.1 ml and a 50% inhibitory concentration (IC50) to GCV of 5.847 µmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance.

CONCLUSIONSPhenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.

Antiviral Agents ; pharmacology ; Cell Line ; Cytomegalovirus ; drug effects ; genetics ; isolation & purification ; Drug Resistance, Viral ; genetics ; Ganciclovir ; pharmacology ; Genes, Viral ; Genotype ; Hematopoietic Stem Cell Transplantation ; Humans ; Mutation ; Phosphotransferases (Alcohol Group Acceptor) ; genetics

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

BACKGROUNDCytomegalovirus (CMV) retinitis is the most severe intraocular complication that results in total retinal destruction and loss of visual acuity in patients with acquired immunodeficiency syndrome (AIDS). This study aimed to investigate the fundus characteristics, systemic manifestations and therapeutic outcomes of CMV retinitis associated with AIDS.

METHODSIt was a retrospective case series. CMV retinitis was present in 39 eyes (25 patients). Best corrected visual acuities, anterior segment, fundus features, fundus fluorescence angiography (FFA) and CD4(+) T-lymphocyte counts of the patients with CMV retinitis associated with AIDS were analyzed. Intravitreal injections of ganciclovir (400 µg) were performed in 4 eyes (2 patients).

RESULTSRetinal vasculitis, dense, full-thickness, yellow-white lesions along vascular distribution with irregular granules at the border, and hemorrhage on the retinal surface were present in 28 eyes. The vitreous was clear or mildly opaque. Late stage of the retinopathy was demonstrated in 8 eyes characterized as atrophic retina, sclerotic and attenuated vessels, retinal pigment epithelium (RPE) atrophy, and optic nerve atrophy. Retinal detachment was found in 3 eyes. The average CD4(+) T-lymphocyte count in peripheral blood of the patients with CMV retinitis was (30.6 ± 25.3) × 10(6)/L (range, (0 - 85) × 10(6)/L). After intravitreal injections of ganciclovir, visual acuity was improved and fundus lesions regressed.

CONCLUSIONSCMV retinitis is the most severe and the most common intraocular complication in patients with AIDS. For the patients with yellow-white retinal lesions, hemorrhage and retinal vasculitis without clear cause, human immunodeficiency virus (HIV) serology should be performed. Routine eye examination is also indicated in HIV positive patients.

Acquired Immunodeficiency Syndrome ; complications ; immunology ; metabolism ; Adult ; Antiviral Agents ; pharmacology ; CD4-Positive T-Lymphocytes ; metabolism ; Cytomegalovirus Retinitis ; drug therapy ; etiology ; immunology ; metabolism ; Female ; Fluorescein Angiography ; Ganciclovir ; pharmacology ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Young Adult

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 investigate the enhancing effect of all-trans retinoic acid (ATRA) on the bystander effect of the herpes simplex virus thymidine kinase(HSV-TK)/ganciclovir (GCV) against androgen unresponsive prostate cancer.

METHODSThe bystander effect of the HSV-TK/GCV system was measured by methyl thiazolyl tetrazolium (MTT) assay on PC-3 cells before and after ATRA treatment. The growth and the histopathology of transplant tumors were observed in 4 groups of nude mice with prostate cancer.

RESULTSATRA augmented significantly the bystander effect of the HSV-TK/GCV system by reducing TK positive PC-3 cells from 50% to 30% (P < 0.05). HSV-TK showed an inhibiting effect, while ATRA with the HSV-TK/GCV system produced significant effect on prostate cancer 1 week earlier than the former (P < 0.05).

CONCLUSIONATRA can argument the in vivo and in vitro bystander effect of the HSV-TK/GCV system in the treatment of androgen unresponsive prostate cancer.

Animals ; Antineoplastic Agents ; pharmacology ; Bystander Effect ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Humans ; Male ; Mice ; Mice, Nude ; Prostatic Neoplasms ; genetics ; pathology ; therapy ; Reverse Transcriptase Polymerase Chain Reaction ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Tretinoin ; pharmacology ; Xenograft Model Antitumor Assays ; methods

PURPOSE: We have used a genetically attenuated adenoviral vector which expresses HSVtk to assess the possible additive role of suicidal gene therapy for enhanced oncolytic effect of the virus. Expression of TK was measured using a radiotracer-based molecular counting and imaging system. MATERIALS AND METHODS: Replication-competent recombinant adenoviral vector (Ad-deltaE1B19/55) was used in this study, whereas replication-incompetent adenovirus (Ad-deltaE1A) was generated as a control. Both Ad-deltaE1B19/55-TK and Ad-deltaE1A-TK comprise the HSVtk gene inserted into the E3 region of the viruses. YCC-2 cells were infected with the viruses and incubated with 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (I-131 FIAU) to measure amount of radioactivity. The cytotoxicity of the viruses was determined, and gamma ray imaging of HSVtk gene was performed. MTT assay was also performed after GCV treatment. RESULTS: On gamma counter-analyses, counts/minute (cpm)/microgram of protein showed MOIs dependency with deltaE1B19/55-TK infection. On MTT assay, Ad-deltaE1B19/55-TK led to more efficient cell killing than Ad-deltaE1A-TK. On plate imaging by gamma camera, both Ad-deltaE1B19/55-TK and Ad-deltaE1A-TK infected cells showed increased I-131 FIAU uptake in a MOI dependent pattern, and with GCV treatment, cell viability of deltaE1B19/55-TK infection was remarkably reduced compared to that of Ad-deltaE1A-TK infection. CONCLUSION: Replicating Ad-deltaE1B19/55-TK showed more efficient TK expression even in the presence of higher-cancer cell killing effects compared to non-replicating Ad-deltaE1A-TK. Therefore, GCV treatment still possessed an additive role to oncolytic effect of Ad-deltaE1B19/55-TK. The expression of TK by oncolytic viruses could rapidly be screened using a radiotracer-based counting and imaging technique.
Adenoviridae/*genetics/physiology ; Cell Line, Transformed ; Cell Line, Tumor ; Ganciclovir/pharmacology ; Gene Expression ; Gene Therapy/methods ; Genetic Vectors ; Humans ; *Oncolytic Virotherapy ; Oncolytic Viruses/*genetics/physiology ; Simplexvirus/genetics ; Tetrazolium Salts/analysis ; Thiazoles/analysis ; Thymidine Kinase/*genetics/metabolism ; Transgenes ; Viral Proteins/*genetics/metabolism ; Virus Replication

To make a universal gene targeting vector fitting for most gene and delete positive selection gene after targeting successfully, a vector named pA2T was constructed by inserting one neomycin gene (neo) for positive selection and two same herpes simplex virus thymidine kinase gene HSV-tk1 and HSV-tk2 for negative selection into the vector of pGEM-3Z, and two locus of crossing-over (x) in P1 (LoxP) and two different multiple cloning sites (MCS) were inserted into two flanks of neo separately. There were eight rare cloning sites between neo and HSV-tk1 and five rare cloning sites between neo and HSV-tk2, and neo, HSV-tk1 and HSV-tk2 could be translated respectively in the pA2T. Transfection of the pA2T into goat fetus fibroblast cells with Lipofectamine 2000 conferred resistance to geneticin (G418) and resistance to ganciclovir (GAC) in the cells, which suggested the positive and negative selectable markers could express in the cells and thus the vector pA2T could be used as a universal gene targeting vector. Transformation of the pA2T into the BM25.8 expressing Cre recombinase conferred neo was deleted in the pA2T, which suggested the LoxP was active. Thus, this vector can be inserted by most gene sequences as homologous sequences and positive selection gene can be deleted after targeting successfully, which is very convenience for the production of transgenic animals using gene targeting method.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Molecular ; Ganciclovir ; pharmacology ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Gentamicins ; pharmacology ; Goats ; Integrases ; genetics ; Neomycin ; pharmacology ; Phosphotransferases ; genetics ; metabolism