Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.
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PMID:Efficient gene delivery into epstein-barr virus (EBV)-transformed human B cells mediated by replication-defective herpes simplex virus-1 (HSV-1): A gene therapy model for EBV-related B cell malignancy. 983 67

We and others have proposed mammalian cells as gene delivery vehicles with the potential for overcoming physiological barriers to viral vectors. To that end, we previously have shown the potential of CD34+ endothelial progenitors for systemic gene delivery in a primate angiogenesis model. Here we seek to explore the utility of CD34+ cells of human origin as vehicles for toxin genes and, in particular, to measure their capacity to effect a cytotoxic bystander effect in human endothelium and tumor cells. To this end, CD34+ cells were transduced with TOZ.1, a nonreplicative herpes simplex vector encoding thymidine kinase. To test the capacity of CD34+ cells to induce a cytotoxic bystander effect in target cells, we performed mixing experiments, whereby TOZ.1-transduced CD34+ cells were mixed with either human vascular endothelial cells or human ovarian tumor cells (SKOV3.ip1). Cell viability was measured by the MTS assay. Lastly, mixtures of TOZ.1-transduced CD34+ cells and SKOV3.ip1 tumor cells were injected s.c. to evaluate the bystander effect in vivo. After transduction of CD34+ cells with TOZ.1, treatment with ganciclovir induced the killing of 99% of cells. In cell-mixing experiments, a linear correlation was observed between the percentages of TOZ.1-transduced CD34+ cells and total cell killing. For example, when 50% of CD34+ transduced cells were mixed with nontransduced SKOV3.ip1, >70% of all cells died. Similarly, when the same percentage was mixed with human vascular endothelial cells, >80% of the total number of cells died. In vivo studies showed an abrogation of tumor formation when TOZ.1-transduced CD34+ cells and ganciclovir were administered. Our observations establish the feasibility of a method for cell-based toxin gene delivery into disseminated areas of tumor angiogenesis.
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PMID:Genetically modified CD34+ cells exert a cytotoxic bystander effect on human endothelial and cancer cells. 1110 65

Dunning R3327 AT-1 rat prostate tumor cells were transfected with a double-fusion suicide gene (CDglyTK) that coded for the cytosine deaminase from E. coli and the thymidine kinase (TK) from HSV-1. The resulting cell line AT-1/CDglyTK was incubated with 10 and 20 microg/ml 5-FC or 0.25 microg/ml GCV, or both 5-FC and GCV 96 hours before harvest. The MTS assay detected cell viabilities of 50+/-5 and 25+/-5% after 5-FC treatment, and 50+/-5% after GCV treatment. The dye exclusion and the colony-forming assay confirmed the data of the MTS assay with GCV (47+/-5 and 32+/-5%), but presented different results for the 5-FC incubation. We detected 100+/-1 and 85+/-5% viable cells after 10 microg/ml 5-FC, and 97+/-1 and 85+/-5% after 20 microg/ml 5-FC treatment, respectively. S-phase arrest in both suicide gene systems was noticeable and a significant increase in cell granularity was observed after incubation with GCV or GCV & 5-FC. This study demonstrates that 5-FC and the metabolized 5-FU act not only as genotoxic reagents, but also as RNA-directed agent, because of the recovery of the cells. On the other hand, a significant S-phase block could be observed after 24 hours incubation with GCV. This short time is enough to incorporate the genotoxic GCV metabolites in the nascent DNA to impair the cell cycle.
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PMID:Comparison of different methods to assess the cytotoxic effects of cytosine deaminase and thymidine kinase gene therapy. 1467 73

Hypoxia-inducible factors, key transcription factors for hypoxia-dependent gene expression, play important roles in angiogenesis and tumor growth. The VHL protein binds to the alpha subunit of (HIF-alpha) for its oxygen-dependent degradation. VHL mutations are found frequently in sporadic RCC. Disruption of VHL results in an abnormal accumulation of HIF-alpha, leading to the upregulation of downstream genes such as the vascular endothelial growth factor gene. We constructed a luciferase reporter vector driven by hypoxia-responsive elements (5HRE/luc) and a therapeutic vector expressing a herpes simplex virus thymidine kinase gene (5HRE/tk). In the transient transfection assay using VHL-deficient 786-O cells, constitutive luciferase expression was detected under both aerobic and hypoxic conditions. In contrast, 786-O cells transfected with a wild-type VHL showed hypoxia-inducible luciferase activity. In in vitro MTS assay, 50% of growth inhibition of 786-O cells stably transfected with 5HRE/tk was achieved with exposure to 0.2 microg/mL of GCV under both aerobic and hypoxic conditions. Xenografts of the stable clone in SCID mice exhibited a marked regression on daily injections of GCV (50 mg/kg) for 10 days. In conclusion, a hypoxia-responsive vector may have therapeutic potential for RCC with VHL mutations.
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PMID:A tumor-specific gene therapy strategy targeting dysregulation of the VHL/HIF pathway in renal cell carcinomas. 1590 70

Gene therapy with herpes simplex virus thymidine kinase gene (HSV-TK), which is also known as "suicide" gene therapy, is effective in various tumor models. The lack of a safe and efficient gene delivery system has become a major obstacle to "suicide" gene therapy. In this study, the cytotoxicity and transfection efficiency of graphene oxide-hydroxyapatite (GO-Hap) were analyzed by MTS and flow cytometry, respectively. A series of assays were performed to evaluate the effects of GO-HAp/p-HRE/ERE-Sur-TK combined with ganciclovir treatment on growth of human breast normal and cancer cells. The results showed that GO-HAp nanocomposites effectively transfected cells with minimum toxicity. GO-HAp/p-HRE/ERE-Sur-TK combined with ganciclovir treatment inhibited the proliferation and induced cell apoptosis in cancer cells, while the cytotoxic effects are tolerable in normal breast cells. We conclude that the GO-HAp nanocomposites have significant potential as a gene delivery vector for cancer therapy.
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PMID:Graphene oxide-hydroxyapatite nanocomposites effectively deliver HSV-TK suicide gene to inhibit human breast cancer growth. 3009 97

The aim of this study was to develop an interventional oncologic technique, "Image-guided intratumoral radiofrequency hyperthermia (RFH)-enhanced herpes simplex virus-thymidine kinase (HSV-TK) gene therapy of ovarian cancer. This study consisted of three portions: (1) serial in-vitro experiments to establish "proof-of-principle" of this novel technique using human ovarian cancer cells; (2) serial in-vivo experiments to validate technical feasibility using animal models with the same orthotopic ovarian cancers; and (3) serial investigations into the underlying bio-molecular mechanisms of this technique. We included four subject groups: (i) combination therapy with RFH+HSV-TK gene therapy; (ii) gene therapy-only; (iii) RFH-only; and (iv) Phosphate-buffered saline (PBS). For in-vitro experiments, confocal microscopy and MTS assays were performed to quantify HSV-TK gene expression and assess cell viability. For in-vivo experiments, bioluminescence optical and ultrasound imaging were used to assess therapeutic effectiveness. These results were correlated with subsequent pathologic/laboratory studies to further elucidate the biologic mechanisms of this technique. In in-vitro experiments, combination therapy resulted in the lowest cell proliferation and greatest increase in HSV-TK gene expression among four subject groups. In in-vivo experiments, combination therapy lead to significant decreases of bioluminescence signals and sizes of tumors in combination therapy by optical and ultrasound imaging. Pathology/laboratory examinations confirmed the significantly increased expression of Bax, Caspase-3, HSP70, IL-2, and CD94 in cancer tissues subjected to combination therapy. "Image-guided intratumoral RFH-enhanced direct gene therapy" is an effective interventional oncologic technique which functions through apoptotic/anti-tumor immunity pathways. This technical development may open new avenues for treating ovarian cancer.
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PMID:Epithelial ovarian cancer: feasibility of image-guided intratumoral radiofrequency hyperthermia-enhanced direct gene therapy. 3090 35