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

Infection of rat RT2 glioma cells in vitro with an adenovirus (ADV-TK) expressing herpes simplex virus (HSV) thymidine kinase (TK) and subsequent exposure to 5-bromo-2'-deoxycytidine (BrdC), which is specifically incorporated into ADV-TK-infected cell DNA as 5-bromo-2'-deoxyuridine (BrdU), results in significant radiosensitization (sensitizer enhancement ratio: 1.4-2.3) compared with Ad beta gal-infected cells. Cell killing correlated well with increased BrdU DNA incorporation and with apoptosis. Whereas radiation (4 Gy) alone was relatively ineffective in inducing apoptosis, treatment with HSV-TK/BrdC resulted in BrdC dose- (10-100 microM) and time-dependent (24-48 hours) increases, and the combination of the two treatments produced a synergistic response (1.5- to 2-fold). To investigate the effects of the ADV-TK/BrdC treatment in vivo, RT2 cells were grown as soft tissue tumors in Fischer 344 rats and conditions for virus infusion were optimized by altering the volume and rate of infusion using a rate-controlled positive pressure device. We found that relatively large volumes (100-150 microL) of virus delivered at rates of < or = 1 microL/minute were optimal and gave uniform and reproducible results. Using these optimal infusion conditions, we were able to achieve 40% adenovirus infection in the tumor. Infection of RT2 tumors with ADV-TK and continuous administration of BrdC from an osmotic pump resulted in significant (.001 < P < .009) tumor regression 6 days after radiation (30 Gy delivered as 2 x 5 Gy over 3 days) compared with controls. In situ staining of sectioned tumors with anti-BrdU antibody or by high-performance liquid chromatography analysis of extracted and hydrolyzed tumor DNA confirmed that we obtained efficient and specific incorporation of BrdU into tumor cells. These results suggest that adenovirus-mediated delivery of HSV-TK in combination with BrdC and radiation can potentially be an efficient combination modality for the treatment of gliomas.
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PMID:Radiosensitization of rat glioma with bromodeoxycytidine and adenovirus expressing herpes simplex virus-thymidine kinase delivered by slow, rate-controlled positive pressure infusion. 1083 Jul 25

Cellular cytoplasmatic thymidine kinase 1 (TK1) catalyzes the intracellular phosphorylation of anti-HIV-1 nucleoside analogs zidovudine (AZT) and stavudine (d4T) to the corresponding monophosphate form. In HIV-1-infected patients, treated with combination therapy including one of these compounds for more than 1 year, enzymatic activity of TK1 in peripheral blood mononuclear cells (PBMC) was determined by radioactive assay. TK1 activity in PBMC of HIV-1-infected patients correlated with CD4 cell count (r = 0.4, p<0.05) and HIV-1 RNA copy number (r = 0.4, p<0.05), being lower in patients with decreased CD4 cell count and high viral load. Furthermore,TK1 activity differs between HIV-1-infected individuals treated for more than 6 months (13.5 pmol/mg/h) compared to patients treated for less than 6 months (28.1 pmol/mg/h; p<0.05) with chemotherapeutic agents including thymidine analogs. The results demonstrate that TK1 deficiency in PBMC of HIV-1 infected patients may develop due to continuous treatment with thymidine analogs and correlates with a more progressed stage of disease expressed as diminished CD4 cell count and increased viral load.
Infection
PMID:Activity of cellular thymidine kinase 1 in PBMC of HIV-1-infected patients: novel therapy marker. 1096 25

Gene therapy using the herpes simplex virus thymidine kinase (HSV-TK) gene in combination with the drug ganciclovir (GCV) is a promising approach for the treatment of cancer-inducing gliomas, a tumor with a poor prognosis. In an attempt to limit the toxic effects on normal tissues, we constructed a recombinant adenoviral vector, Adgfa2TK, in which the HSV-TK gene is driven by the promoter for the gene encoding glial fibrillary acidic protein, an intermediate filament protein expressed primarily in astrocytes. Infection by Adgfa2TK of a glial cell line (C6) and a non-glial cell line (MDA-MB-231) revealed markedly increased expression of HSV-TK in glial cells as determined by Western blot. In comparison, high HSV-TK protein levels were produced in both cell lines after infection with a control virus, AdCMVTK, in which the constitutive cytomegalovirus viral promoter was used to direct HSV-TK expression. Infection of two glial cell lines (C6, U251) and two non-glial cell lines (HepG2, MDA-MB-231) with Adgfa2TK followed by GCV treatment revealed high toxicity in glial cell lines (50% growth inhibitory concentration: <2 microg/mL of GCV) with little or no toxicity (50% growth inhibitory concentration: >75 microg/mL) in the non-glial cell lines. In vivo, injection of Adgfa2TK into C6 tumors grown in nude mice followed by intraperitoneal GCV treatment significantly repressed tumor growth compared with the controls. Adgfa2TK may be useful for directing expression of the HSV-TK gene to gliomas.
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PMID:Inhibition of glioma cells in vitro and in vivo using a recombinant adenoviral vector containing an astrocyte-specific promoter. 1097 72

Expression of suicide genes (e.g. herpes simplex virus thymidine kinase,HSV-TK) in T cells is an appealing approach to regulate graft-versus-host disease in adoptive immunotherapy. Here we report the optimization of retroviral infection of canine T cells. Canine T cells were stimulated either with phytohemagglutinin (PHA, 2 microg/ml) for 24-72 hours or with 100 U/ml interleukin-2 for seven days. Stimulated cells were co-cultivated with irradiated virus-producing cells. Transduction efficiencies ranged from 4% to 45% using PG13, a gibbon ape leukemia virus envelope (env) pseudotyped packaging cell line. Infection of cells with GPenvAM12, expressing the amphotropic Moloney murine leukemia virus env, did not yield a satisfactory percentage of transduced cells. Enrichment of transduced cells was performed using immunoselection, and gave a purity of up to 98%. Transfusion of 1 x 10(6) transduced cells per kilogram body weight showed that transduced cells could convert mixed chimerism to 100% and transfer immunity to a specific antigen. Transduced cells were repeatedly detected in peripheral blood and bone marrow by polymerase chain reaction with primers specific for the HSV-TK gene. We have demonstrated the feasibility of using the canine model to study gene therapy as a preclinical model.
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PMID:Expression of HSV-TK suicide gene in primary T lymphocytes: the dog as a preclinical model. 1097 36

The Hc fragment of tetanus toxin (Hc) retains the specific nerve cell binding and transport properties of the holotoxin, but lacks any toxicity. We are investigating the potential for utilising its neurotropism for targeted gene delivery to the central nervous system. Previously we reported the use of Hc-polylysine conjugates for selective gene transfer into neuronal cells in vitro. However, as attempts to apply these constructs in vivo were not successful, we have extended these studies to modification of the tropism of adenoviral vectors. Either Hc-polylysine conjugates or the Fab fragment of a neutralising anti-knob antibody covalently bound to Hc were attached to the virus. Infection of neuronal and non-neuronal cell lines with retargeted virus showed highly increased neuronal cell selectivity, but no significant enhancement of gene delivery into these cells. High concentrations of free Hc blocked the infectivity of the retargeted vector efficiently. Intramuscular injection of retargeted virus into mouse tongues resulted in selective gene transfer to the neurons of the hypoglossal nucleus, where no pathological changes were observed. As differentiated neurons do not undergo cell division, appropriate vectors carrying a thymidine kinase gene, which allows selective elimination of dividing cells, may be exploitable for the treatment of tumours of the central nervous system. The demonstrated suitability of the Hc fragment of tetanus toxin as targeting moiety for viral vectors also indicates a potential for gene therapy of inherited neurodegenerative diseases such as spinal muscular atrophy.
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PMID:Retargeting of adenoviral vectors to neurons using the Hc fragment of tetanus toxin. 1102 97

The EGFP-tk retroviral vector, encoding enhanced green fluorescent protein (EGFP) and the herpes simplex virus thymidine kinase (HSV-tk) packaged in a Phoenix amphotropic cell line, was used to transduce healthy donor T lymphocytes. Infection yielded a mean of 41.8 +/- 9.3% SD (range 31.1-48.4%) EGFP-positive cells and a mean of 92 +/- 2% SD (range 90-94%) after cell sorting. EGFP expression remained stable for 30 d after infection. The entire gene transfer procedure had no significant effect on lymphocyte subsets and slightly reduced clonogenicity. Ganciclovir (gcv) treatment (1 microg/ml x 10 d) killed all EGFP-positive cells in the transduced and transduced/sorted populations, but had no effect on untransduced controls. Our results show that primary T lymphocytes can be transduced using an EGFP-tk vector that yields a homogeneous infected population without affecting lymphocyte subsets, function and clonogenicity.
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PMID:Retrovirus-mediated transfer of the herpes simplex virus thymidine kinase and enhanced green fluorescence protein genes in primary T lymphocytes. 1105 80

Infection of mouse trigeminal ganglia by herpes simplex virus induces cytokine expression that persists long after infectious virus or viral antigens become undetectable. To examine mechanisms underlying this phenomenon, we used a thymidine kinase mutant, dlsptk, which fails to replicate in ganglia and does not reactivate upon ganglionic explant. Using quantitative reverse transcriptase-polymerase chain reaction assays, we found that levels of interferon-gamma and tumor necrosis factor-alpha transcripts in dlsptk-infected ganglia were lower than those in wild type-infected ganglia, but were significantly (eight- to 10-fold) higher than those in mock-infected ganglia from Day 3 to Day 100 postinfection. We also studied latency-associated transcript (LAT) negative mutants that exhibit increased expression of productive cycle transcripts in ganglia. Ganglia infected with these mutants contained levels of cytokine transcripts similar to those in wild type-infected ganglia; any increases in viral antigen expression mediated by the LAT deletion were not accompanied by increased cytokine expression. Thus, neither viral replication, the ability to reactivate, nor LAT expression in ganglia is required for persistent elevated cytokine expression. The results provide indirect evidence that low-level expression of viral productive cycle genes in neurons can provide signals that elicit cytokine expression.
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PMID:Persistent elevated expression of cytokine transcripts in ganglia latently infected with herpes simplex virus in the absence of ganglionic replication or reactivation. 1111 95

We have demonstrated previously the oncolytic effects of a systemically delivered, replicating vaccinia virus. To enhance the tumor specificity of this vector, we have developed a combined thymidine kinase-deleted (TK-) and vaccinia growth factor-deleted (VGF-) vaccinia virus and investigated its properties in vitro and in vivo. The gene for enhanced green fluorescent protein (EGFP) was inserted into the TK locus of a VGF- vaccinia virus by homologous recombination creating a double-deleted mutant vaccinia virus (vvDD-GFP). Infection of resting and dividing NIH3T3 cells with vvDD-GFP yielded reduced viral recovery compared with wild-type (WT), TK-, or VGF- viruses from resting cultures but equivalent virus recovery from dividing cultures. Eight days after nude mice were injected i.p. with 10(7) plaque-forming units (pfu) of WT, TK-, VGF-, or vvDD-GFP vaccinia virus, tissues and tumor were harvested for viral titer determination. No virus was recovered from the brains of mice injected with vvDD-GFP compared with the other viruses, which ranged from 130 to 28,000 pfu/mg protein; however, equivalent amounts were recovered from tumor. There was no toxicity from vvDD-GFP because nude mice receiving 10(8) pfu of IP vvDD-GFP lived >100 days, whereas mice receiving WT, VGF-, or TK- virus had median survivals of only 6, 17, and 29 days, respectively. Similar results were seen when 10(9) pfu of vvDD-GFP were given. Nude mice bearing s.c. murine colon adenocarcinoma (MC38) had significant tumor regression after treatment with 10(9) pfu of systemic (i.p.) vvDD-GFP compared with control (mean tumor size, 180.71 +/- 35.26 mm(3) versus 2796.79 +/- 573.20 mm(3) 12 days after injection of virus). Our data demonstrate that a TK- and VGF- mutant vaccinia virus is significantly attenuated in resting cells in vitro and demonstrates tumor-specific replication in vivo. It is a promising vector for use in tumor-directed gene therapy, given its enhanced safety profile, tumor selectivity, and the oncolytic effects after systemic delivery.
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PMID:Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. 1175 95

Infection of the mouse trigeminal ganglia (TG) is the most commonly used model for the study of herpes simplex virus type 1 (HSV-1) latency. Its popularity is caused, at least in part, by the perception that latent infection can be studied in this system in the absence of spontaneous viral reactivation. However, this perception has never been rigorously tested. To carefully study this issue, the eyes of Swiss-Webster mice were inoculated with HSV-1 (KOS), and 37-47 days later the TG were dissected, serial-sectioned, and probed for HSV-1 ICP4, thymidine kinase, glycoprotein C, and latency-associated transcript RNA by in situ hybridization. Serial sections of additional latently infected TG were probed with HSV-1-specific polyclonal antisera. Analysis of thousands of probed sections revealed abundant expression of viral transcripts, viral protein, and viral DNA replication in about 1 neuron per 10 TG tested. These same neurons were surrounded by a focal white cell infiltrate, indicating the presence of an antigenic stimulus. We conclude that productive cycle viral genes are abundantly expressed in rare neurons of latently infected murine TG and that these events are promptly recognized by an active local immune response. In the absence of detectable infectious virus in these ganglia, we propose the term "spontaneous molecular reactivation" to describe this ongoing process.
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PMID:Spontaneous molecular reactivation of herpes simplex virus type 1 latency in mice. 1177 30

Nasopharyngeal carcinoma (NPC) is a human cancer of epithelial cell origin. Infection by Epstein-Barr virus has been shown to be closely associated with this tumor. Recent studies have indicated that another common epitheliotropic virus, human papillomavirus (HPV), is also found in a significant number of NPC cases. In this study, we evaluated the feasibility of using the HPV regulatory long control region (LCR) to drive the expression of the thymidine kinase (tk) gene to achieve chemosensitivity for gene therapeutic treatment of NPC. Testing HPV-11-LCR-tk constructs in NPC cell lines in the presence of ganciclovir (GCV) led to 50-60% cell death of transfected cells. The therapeutic efficacy was further tested in an in vivo model using nude mice transplanted with tumors derived from transfected NPC cells. Injection of 50 mg/kg body weight GCV twice daily for 14 days resulted in visually complete regression of the transplanted NPC tumor loads within 20 days after GCV treatment. Taken together, results from this pilot study indicate the feasibility of the development of a gene therapeutic protocol based on the chemosensitive gene constructs described in this paper.
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PMID:Induction of chemosensitivity in nasopharyngeal carcinoma cells using a human papillomavirus regulatory sequence and the thymidine kinase gene. 1181 24


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