Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Analysis of factors determining replication of BK virus (BKV) episomal vectors in human cells showed that vector copy number was related to the level of BKV T antigen expression. T antigen was synthesized efficiently, as assessed by indirect immunofluorescence, in vector-transfected primary embryonic fibroblasts undergoing neoplastic transformation. Surprisingly, transfected continuous cell lines (143 B, HeLa and KB), kept under biochemical selection or tested in transient assays, produced negligible amounts or no T antigen, revealed only by a sensitive ELISA test, suggesting that in these cells vector amplification was under the control of cellular factors. Presence or absence of BKV late region sequences, BKV strain, orientation of the inserted genes and presence or absence of selection were not relevant for vector replication. Type of biochemical selection, however, was important, since BKV vectors containing the thymidine kinase gene replicated better than those containing the neo gene. Despite great variability, vector copy number increased in transfected clones of adenovirus 5-transformed 293 cells, in the absence of immunofluorescence detectable T antigen. These cells express adenovirus immediate early proteins E1A and E1B which may directly or indirectly activate BKV origin of replication.
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PMID:Factors affecting amplification of BK virus episomal vectors in human cells. Brief report. 283 49

Inhibition of protein synthesis by cycloheximide after transfection and subsequent removal of the drug increased the transformation efficiency of primary cells by plasmids containing the left 4.5, 6.7, or 16% of the adenovirus (Ad) genome. The enhancement factor ranged from 2 to as much as 70 depending on the size of the viral DNA fragments used. Addition of cycloheximide before or at the time of transfection inhibited transformation, suggesting that viral protein synthesis is important during the early phase of transformation. Transient expression assays showed that cells treated with cycloheximide post-transfection contained as much as three times the amount of viral RNA transcribed from regions E1A and E1B. Conversion of a rat cell line lacking thymidine kinase activity (TK-) to the TK+ phenotype by a plasmid containing the herpes TK gene was severely inhibited by the drug treatment, suggesting that the enhancement effects of cycloheximide on transformation may be specific for Ad DNA. Cycloheximide treatment also increased the number of transformants induced by a transformation defective E1B mutant of Ad12 (cyt mutant). Plasmid containing only the E1A region of Ad12 transformed primary rat kidney cells with very low efficiency. The inclusion of E1B in the transfecting DNA fragments increased the transformation frequency by more than 400-fold, much higher than that achieved by cycloheximide. Thus, cycloheximide cannot replace E1B functions in transformation efficiency.
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PMID:Inhibition of protein synthesis enhances transformation of primary cells by viral DNA. 301 45

The level of expression of thymidine kinase (TK), heat shock protein 70 (HSP70), beta-tubulin and p53 was assessed in human embryo kidney cells (HEKs) infected with adenovirus type 12 (Ad 12) and Ad 12 early region 1 (E1) mutants. HSP70, beta-tubulin and p53 levels were unchanged but TK activity was dramatically increased following wild-type infection. The initial activation of TK required the expression of the product of the E1A 13S mRNA but sustained expression only occurred with those viruses expressing the E1B proteins as well. A number of human cell lines transformed with either Ad 12 or Ad 5 E1 DNA were also assessed for the level of expression of HSP70, beta-tubulin and p53. Both HSP70 and beta-tubulin levels were greatly increased compared with primary human cells although there was considerable variation between lines. p53 was only expressed at high levels in Ad 12-transformed lines expressing E1A and E1B proteins.
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PMID:Modulation of the level of expression of cellular genes in adenovirus 12-infected and transformed human cells. 352 49

Mutants dl312, dl314, hr1, and hr3 with mutations in region E1A of adenovirus type 5 were defective for the induction of cell cycle abnormalities detectable by flow cytometry, cell DNA replication, thymidine kinase production, and chromosome aberrations and did not synthesize the viral DNA-binding protein (E2A) in rat cells. dl311, a leaky E1A mutant, induced cell cycle effects at high multiplicity in only one of three experiments, and synthesized the DNA-binding protein. hr7 (E1B) gave a wild-type response in all tests. dl313 was also positive in all tests, although it induced fewer polyploid cells than did wild-type virus, probably because of the leftward extension of the dl313 E1B deletion into E1A. sub315 and sub316, with mutations which also span the E1A-E1B border, synthesized DNA-binding protein, but caused no cell cycle alterations detectable by flow cytometry in rat or mouse cells. Although the participation of other viral early regions cannot be completely excluded, our results suggest that alteration of cell cycle progression is a direct effect of E1A unrelated to its control of other viral early regions, and may be the function of E1A in transformation.
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PMID:Adenovirus-induced alterations of the cell growth cycle: a requirement for expression of E1A but not of E1B. 682 12

The adenovirus type 5 E1A transcriptional control region contains an element with enhancer properties located between -141 and -305 relative to the E1A cap site at +1. The enhancer element is located at or very close to a sequence required in cis for packaging of viral DNA. Deletion of the element reduces both the rate of transcription and steady-state levels of E1A mRNAs in virus-infected cells. Such deletions also cause a modest reduction in activity of the E1B transcription unit, which is located immediately downstream of the E1A unit. The enhancer element includes repeated core sequences. Function is retained when the element is moved to the 3' side of the E1A gene in either possible orientation, and the E1A enhancer functions in cis to enhance transformation by the herpesvirus thymidine kinase gene in both mouse and human cells.
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PMID:The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element. 687 91

Previous work by this group has established that E1-defective, recombinant adenoviruses can be replication-enabled by the codelivery of a plasmid encoding the deleted E1 functions, a strategy now designated conditional replication-enablement system for adenovirus (CRESA). In the studies reported here, the original replication-enabling plasmid was replaced by two separate plasmids that encoded the necessary E1A and E1B functions, respectively. An RNA transcript encoding the requisite E1A functions was shown to substitute functionally for the E1A plasmid without significant loss of new adenovirus production in in vitro experiments. No replication competent adenovirus was detectable in the cells treated with the plasmids, or the RNA and plasmid combinations. Subcutaneous human tumor nodules containing a fraction of cells cotransduced with the replication-enabling RNA + DNA and an adenovirus containing a herpes simplex virus thymidine kinase (HSVtk) expression cassette were reduced to a greater extent than control nodules containing the same fraction of cells cotransduced with the virus and an irrelevant plasmid. These experiments show that an E1-defective adenovirus can be conditionally replication-enabled by an RNA transcript encoding the required E1 functions, and that the replication-enablement is sufficient to produce an augmentation of an adenovirus-mediated therapeutic effect in vivo.
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PMID:E1A RNA transcripts amplify adenovirus-mediated tumor reduction. 904 43

Two obstacles limiting the efficacy of nearly all cancer gene therapy trials are low gene transduction efficiencies and the lack of tumor specificity. Recently, a replication-competent, E1B-attenuated adenovirus (ONYX-015) was developed that could overcome these limitations, because it was capable of efficiently and selectively destroying tumor cells lacking functional p53. In an attempt to improve both the efficacy and safety of this approach, we constructed a similar adenovirus (FGR) containing a cytosine deaminase (CD)/herpes simplex virus type-1 thymidine kinase (HSV-1 TK) fusion gene, thereby allowing for the utilization of double-suicide gene therapy, which has previously been demonstrated to produce significant antitumor effects and potentiate the therapeutic effects of radiation. The FGR virus exhibited the same tumor cell specificity and replication kinetics as the ONYX-015 virus in vitro. Importantly, both the CD/5-FC and HSV-1 TK/GCV suicide gene systems markedly enhanced the tumor cell-specific cytopathic effect of the virus, and, as expected, sensitized tumor cells to radiation. By contrast, neither the FGR virus nor either suicide gene system showed significant toxicity to normal human cells. Both suicide gene systems could be used to suppress viral replication effectively, thereby providing a means to control viral spread. The results support the thesis that the three-pronged approach of viral therapy, suicide gene therapy, and radiotherapy may represent a powerful and safe means of selectively destroying tumor cells in vivo.
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PMID:A novel three-pronged approach to kill cancer cells selectively: concomitant viral, double suicide gene, and radiotherapy. 965 Jun 10

In this study, we evaluated three herpes simplex virus-1 thymidine kinase (HSV-tk) carrying replication-competent adenoviral vectors with and without the Ad5 E1B 55 kDa gene to assess whether this gene product has an influence on their antitumor efficacy, replication kinetics, and potential hepatotoxicity. Furthermore, we assessed the efficacy of these vectors in combination with ganciclovir (GCV). When compared with wild-type adenovirus, the recombinant vectors, in particular the E1B 55 kDa-deleted vector Ad.TK(RC)(II), generated a more efficiently cytopathic effect in proliferating cells, independently of their p53 phenotype. In a s.c. A549 lung cancer xenograft model, the cytoreductive effect of Ad.TK(RC)(II) was enhanced when followed by GCV treatment. In contrast, the efficacy of both E1B 55 kDa-positive vectors could not be further improved by GCV. In an i.p. MDAH 2774 ovarian cancer xenograft tumor model, the survival of animals treated with a prototypical replication-deficient adenovirus expressing HSV-tk (Ad.TK) was improved compared to controls when followed by GCV. In contrast, the cytoreductive efficacy of the replication-competent vectors was diminished when combined with the virostatic GCV. However, the antitumor effect of all replication-competent vectors was superior to combination chemotherapy with paclitaxel and carboplatin. In both tumor models, the oncolytic effect of the E1B 55 kDa-positive vectors was greater than that of Ad.TK(RC)(II). In an attempt to assess the toxicity of these vectors in a nonpermissive host, the viruses were administered systemically to immunocompetent and immunodeficient mice. Greater hepatotoxicity was seen with i.v. administration of the replication-competent viruses than with Ad.TK and in immunocompetent hosts, suggesting involvement of the immune system in the induction of tissue damage. The E1B 55 kDa gene had no significant influence on the liver toxicity of the vectors in this system. At therapeutic doses, intratumoral or i.p. injection of all vectors was well tolerated. Importantly, these replication-competent HSV-tk-expressing vectors were highly susceptible to GCV, representing an effective fail-safe mechanism to abolish viral replication in a clinical setting. Controllable intratumoral viral replication holds promise as a new treatment modality for cancer.
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PMID:The role of the E1B 55 kDa gene product in oncolytic adenoviral vectors expressing herpes simplex virus-tk: assessment of antitumor efficacy and toxicity. 1094 25

Tumor cells that express a fusion gene of Escherichia coli cytosine deaminase (CD) and herpes simplex virus type 1 thymidine kinase (TK) sequences activate and are subsequently killed by the nontoxic prodrugs 5-fluorocytosine and ganciclovir. We have previously developed a recombinant adenovirus containing the CD-TK fusion gene controlled by the human inducible heat shock protein 70 promoter so that heat at 41 degrees C for 1 hour induces therapeutic gene expression. This adenovirus effectively transduces heat-inducible expression of the CD-TK gene into human prostate carcinoma cells. However, because a limited number of cells in a tumor can actually be infected, we created a replicating adenoviral vector to increase CD-TK gene expression. This vector is a replication-competent, E1B-attenuated adenoviral vector containing the hsp70 promoter-driven CD-TK gene (Ad.E1A(+)HS-CDTK). When human prostate adenocarcinoma DU-145 cells (mutant p53) were infected with the virus at a multiplicity of infection (MOI) of 1 or 10, the viral replication was detected within 2 days at both MOIs. Similar results were observed in human colorectal carcinoma CX-1 cells. When DU-145 cells were infected with the virus at an MOI of 10, incubated for 24 hours, heated at 41 degrees C for 4 hours, and then harvested 20 hours later, Western blot analysis demonstrated that this virus successfully produced viral E1A proteins and heat shock stimulated the CD-TK gene expression by 12.3-fold. In addition, Ad.E1A(+)HS-CDTK effectively suppressed cell proliferation by viral cytopathic effect). Unlike with a replication-incompetent virus (Ad.HS-CDTK), the cytopathic effect of the virus and cytotoxicity in the presence of the prodrugs were still observed even at low MOI (MOI=1.0).
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PMID:Replicating adenoviral vector-mediated transfer of a heat-inducible double suicide gene for gene therapy. 1149 59

Combination therapy with replicative oncolytic viruses is a recent topic in innovative cancer therapy, but few studies have examined the efficacy of oncolytic adenovirus plus replication-deficient adenovirus carrying a suicide gene. We aim to evaluate whether an E1A, E1B double-restricted oncolytic adenovirus, AxdAdB-3, can improve the efficacy for gallbladder cancers (GBCs) of the replication-deficient adenovirus-based herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) therapy directed by the carcinoembryonic antigen (CEA) promoter. Cytopathic effects of AxdAdB-3 plus AxCEAprTK (an adenovirus expressing HSVtk directed by CEA promoter) or AxCAHSVtk (an adenovirus expressing HSVtk directed by a nonspecific CAG promoter) with GCV administration were examined in several GBC lines and normal cells. Efficacy in vivo was tested in severe combined immunodeficiency disease mice with GBC xenografts. Addition of AxdAdB-3 (1 multiplicity of infection, MOI) significantly enhanced the cytopathic effects of AxCEAprTK (10 MOI)/GCV on GBC cells. The augmented effect was attributable to the replication of the AxCEAprTK and also to the enhanced CEA promoter activity, which was presumably transactivated by E1A. In normal cells, AxdAdB-3 (20 MOI) plus AxCEAprTK (200 MOI)/GCV was not cytopathic, whereas AxdAdB-3 (1 MOI) plus AxCAHSVtk (10 MOI)/GCV was significantly toxic. Low-dose AxdAdB-3 (2 x 10(7) PFU, plaque-forming unit) plus AxCEAprTK (2 x 10(8) PFU)/GCV significantly suppressed the growth of GBC xenografts as compared with either AxdAdB-3 (2 x 10(7) PFU)/GCV or AxCEAprTK (2 x 10(9) PFU)/GCV alone. E1A, E1B double-restricted replicating adenovirus at low dose significantly augmented the efficacy of CEA promoter-directed HSVtk/GCV therapy without obvious toxicity to normal cells, suggesting a potential use of this combination for treating GBC and other CEA-producing malignancies.
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PMID:E1A, E1B double-restricted replicative adenovirus at low dose greatly augments tumor-specific suicide gene therapy for gallbladder cancer. 1881 10


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