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)

We have previously demonstrated that genetically modified syngeneic murine tumor cells (KBALB) expressing the herpes simplex virus thymidine kinase gene (HSV-STK) can kill nearby unmodified tumor cells in the presence of ganciclovir (GCV). The killing was mediated by a 'bystander effect' as evidenced by the prolonged animal survival when syngeneic HSV-TK gene-modified tumor cells were inoculated into mice with an intraperitoneal tumor. In this study we investigated whether irradiated xenogeneic HSV-TK gene-modified tumor cells, a human colon carcinoma cell line (HCT) transfected with the HSV-TK gene, can mediate the 'bystander effect' when used in vitro and in vivo. In vitro experiments indicate that irradiated HSV-TK gene-modified xenogeneic cells (HCT) can mediate a bystander effect on the adjacent cells when the tumor population consisted of as few as 10% of the HSV-TK expressing HCT tumor cells. In vivo, animal survival experiments demonstrate that the xenogeneic gene-modified tumor cells could generate the 'bystander effect' in mice with intraperitoneal tumors as evidenced by prolonged animal survival. In addition, histologic examination of the tumors from experimental animals showed extensive tumor necrosis 3 days post HSV-TK/GCV treatment in comparison to control animals. To evaluate the cause of necrosis in vivo, we assayed for cytokines, which may be involved in mediating this process, by performing RT-PCR and immunohistochemistry on tumor RNA and tumor cells, respectively. Production of IL-1 alpha and IL-6 mRNA within the experimental tumors was observed by RT-PCR. However, mRNA expression for other cytokines including IFN-gamma, IL-2 and IL-4 was not present. Immunohistochemical analysis for IL-1 alpha protein showed reactivity within the infiltrating mononuclear cells indicating the release of this soluble factor. These results indicate that the bystander effect can be generated using irradiated xenogeneic cells both, in vitro and in vivo. Furthermore, this process is mediated by the release of cytokines such as IL-1 alpha, IL-6 which enhances the bystander effect in vivo by immunostimulation.
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PMID:The role of cytokines in mediating the bystander effect using HSV-TK xenogeneic cells. 760 May 27

Stem cell factor (SCF) is known to act synergistically with other hematopoietic factors in increasing the colony formation of hematopoietic progenitor cells. We have shown that interleukin-3 (IL-3)-dependent proliferation of NFS-60 cells is associated with the induction of a specific calmodulin-binding protein of about 68 kD (CaM-BP68). To evaluate the relationship between proliferative stimulation and the induction of CaM-BP68 by cytokines, we examined whether the increased proliferative potential of NFS-60 cells in response to SCF is reflected in an increased induction of the CaM-BP68. We observed that SCF alone has a limited effect on proliferative stimulation and on the induction of CaM-BP68 in factor-deprived NFS-60 cells. However, when combined with IL-3, granulocyte colony-stimulating factor (G-CSF), or IL-6, it caused a significant increase in cytokine-dependent proliferative stimulation, as well as in the induction of CaM-BP68. Furthermore, an increase in IL-3-dependent induction of CaM-BP68 in the presence of SCF coincided with a corresponding increase in thymidine kinase activity, whose expression is linked to G1/S transition of the cells. At low concentrations SCF caused a synergistic increase in IL-3-dependent induction of both CaM-BP68 and thymidine kinase activity. In contrast to the changes in CaM-BP68 and thymidine kinase activity, no significant changes in DNA polymerase alpha were observed in factor-deprived NFS-60 cells in response to IL-3 and/or SCF. These observations suggest an increased expression of CaM-BP68 and thymidine kinase are associated with the synergistic effect of SCF on factor-dependent proliferation of hematopoietic progenitor cells.
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PMID:Stem cell factor enhances interleukin-3 dependent induction of 68-kD calmodulin-binding protein and thymidine kinase activity in NFS-60 cells. 860 34

Lung cancer is a leading cause of cancer death and standard chemotherapies are resulting in only marginal improvements in outcome. Experimental approaches involving gene therapy are attractive in this clinical setting. There are two basic types of genes utilized, either those intended to induce immunity or those that are directly tumoricidal. Immunity-inducing genes that have been used in model (and some human) systems include MHC molecules, costimulatory molecules, and cytokines such as IL-2, IL-4, IL-6, GM-CSF. These are intended to induce effective systemic immune responses against tumor antigens which would not otherwise develop. Direct toxic approaches include the reintroduction of tumor suppressor genes or enzymes which convert non-toxic drugs to toxic ones, such as herpes thymidine kinase. As a means for gene delivery, retroviruses are the most common vehicle, although Adenovirus vectors and direct DNA delivery have specific advantages.
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PMID:Gene therapy for lung cancer. 861 19

In a group of 70 patients with multiple myeloma (MM), formed by 25 patients examined while establishing the diagnosis and 45 patients examined in different stages of the disease, the authors evaluated the relationship of the bromodeoxyuridine "labelling index" (BrdUrd-LI) of myeloma plasma cells assessed by the method of double immunofluorescence (using antibody BU-1) and selected laboratory indicators of the disease. In the whole group the median and mean values of BrdUrd-LI of myeloma plasma cells were 2.0 (0.6-4.4%) and 2.1 +/- 0.9%, in the group of 25 patients examined during diagnosis it was 1.8 (0.6-4.1%) and 1.9 +/- 0.9%, in the group of 45 patients examined during different stages of MM it was 2.4 (0.6-4.4%) and 2.4 +/- 0.8%. Neither in the whole group nor in the sub-groups any statistically significant correlations were found between BrdUrd-LI values and the degree of anaemia, values of S-creatinine, S-MIG, S-albumin, S-B2M, S-ferritin, S-thymidine kinase, S-IL-6, S-IL-2, S-kIL-2R, the percentage ratio of myeloma plasma cells in bone marrow and the synthetic index of myeloma plasma cells paraprotein.
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PMID:[The bromodeoxyuridine index in multiple myeloma. I. Relation with selected laboratory indicators of the disease]. 892 21

The ability to direct expression of genes to astrocytes in mice has been one of the major motivators of transcriptional analyses of the glial fibrillary acidic protein (GFAP) gene. Another has been the possibility of discovering signaling pathways that operate during development, disease, and injury-all states that increase GFAP gene activity-by identifying and working back from the responsible DNA elements. Here we review studies in both these areas and provide practical guidelines for the construction and analysis of GFAP transgenes. Analyses of the GFAP promoter from cell transfection experiments are summarized to provide background information for the studies in transgenics. Another section provides practical information on the construction and analysis of transgenic mice, with particular reference to GFAP transgenes. The survey of analyses of GFAP transcription elements in transgenic mice reveals that a segment of about 2 kb of the 5'-flanking region of the gene is sufficient to direct reporter gene activity to astrocytes with high specificity. This segment also supports a response to brain injury by upregulation of the activity. Developmentally, the transgene activity is seen by e12.5, several days earlier than GFAP protein or mRNA has been detected. GFAP transcription control regions have already been used to express several proteins in astrocytes to evaluate their biological effects. These proteins include IL-3, IL-6, TGF-beta1, the HIV envelop protein gp120, the MHC Class I Db protein, somatosatin, CNTF, and the herpes simplex virus thymidine kinase. In the future many other GFAP transgenes are expected to be produced, with increasing knowledge of the GFAP regulatory elements promising greater sophistication through promoters that can be regulated, have higher activity, and target activity to particular brain regions.
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PMID:GFAP Transgenic Mice 895 47

Activation of transcription at the nuclear factor interleukin 6 (NF-IL-6) DNA binding motif modulates expression of multiple genes important in host adaptive and developmental mechanisms. Studies showing that hypoxia-induced transcription of IL-6 in cultured endothelial cells was due to transcriptional activation by the NF-IL-6 motif in the promoter (Yan, S.-F., Tritto, I., Pinsky, D., Liao, H., Huang, J., Fuller, G., Brett, J., May, L., and Stern, D. (1995) J. Biol. Chem. 270, 11463-11471) led us to prepare transgenic mice using 115- or 14-base pair regions of the promoter encompassing the NF-IL-6 site ligated to the lacZ reporter gene and the basal thymidine kinase promoter. On exposure to hypoxia or induction of ischemia, mice bearing either of the constructs showed prominent expression of the transgene in lung and cardiac vasculature and in the kidney but not in the liver (parenchyma or vasculature). In contrast, transgenic mice bearing a mutationally inactivated NF-IL-6 site showed no increase in transgene expression in hypoxia. Gel retardation assays revealed time-dependent, hypoxia-enhanced nuclear binding activity for the NF-IL-6 site in nuclear extracts of the heart, lung, and kidney but not in the liver; the hypoxia-enhanced band disappeared on addition of antibody to C/EBPbeta-NF-IL-6. Consistent with the specificity of hypoxia-mediated activation of C/EBPbeta-NF-IL-6, gel retardation assays showed no change in the intensity of the hypoxia-enhanced gel shift band in the presence of excess unlabeled oligonucleotide probes or antibodies related to other transcription factors, including NFkappaB, AP1, cAMP response element-binding protein, SP1, and hypoxia-inducible factor 1. These data indicate that the transcription factor NF-IL-6 is sensitive to environmental oxygen deprivation, and the tissue-specific pattern of gene expression suggests that local mechanisms have an important regulatory effect.
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PMID:Nuclear factor interleukin 6 motifs mediate tissue-specific gene transcription in hypoxia. 902 Jan 46

Direct delivery of the herpes simplex virus thymidine kinase (HSVtk) gene, in combination with the prodrug ganciclovir (GC), has been used for the treatment of localised, inoperable tumours. Several groups have shown that when rodent tumours are ablated in vivo with suicide genes, anti-tumour immunity can also be generated. Hence, this approach may also be useful in treating disseminated disease. Here we have studied the mechanisms associated with this anti-tumour immunity. In B16 HSVtk+ tumours being killed in vivo with GC treatment, we observed the induction of a pronounced intratumoural infiltrate of macrophages, CD4+ and CD8+ T cells. In addition, using reverse transcriptase polymerase chain reaction, expression of interleukin (IL)-2, IL-12, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and granulocyte/macrophage colony-stimulating factor (GM-CSF) but not IL-4, IL-6 or IL-10, was observed, a profile of cytokine expression which resembles that of a Th1 immune response. To complement these findings, we also investigated the mechanisms by which expression of HSVtk leads to cell death. Our data show that B16/HSVtk+ cells die predominantly by necrosis, rather than apoptosis, on exposure to GC, a process which may be associated with the generation of anti-tumour inflammatory responses. From these data we propose a model for the induction of anti-tumour immunity using suicide genes and discuss the development of improved vectors for gene therapy to augment these effects in vivo.
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PMID:Generation of an anti-tumour immune response in a non-immunogenic tumour: HSVtk killing in vivo stimulates a mononuclear cell infiltrate and a Th1-like profile of intratumoural cytokine expression. 913 53

In a cancer gene therapy model recombinant adenoviruses expressing the herpes simplex virus thymidine kinase (HSVtk) gene were injected into tumors in situ, either alone or in combination with adenoviruses (Avs) engineered to express IL-2, IL-6 or the costimulatory molecule B7-1. HSVtk phosphorylates the prodrug ganciclovir, thus converting it into an antimetabolite which kills not only HSVtk expressing cells, but also by the 'bystander effect', neighboring untransduced tumor cells. The tumors regressed in 80% of mice upon AvTK/ganciclovir treatment: combinations with AvIL-2, AvIL-6, or AvB7-1 did not improve these results. Cured mice were protected from further challenge with wild-type tumor but not from challenges with an unrelated syngeneic tumor cell line. Since cytotoxic T lymphocyte responses in this tumor model were weak, we analyzed cytokine secretion from spleen cells of treated animals. The best correlate of antitumor immunity in this model was enhanced secretion of GM-CSF, while secretion of IL-2, IL-6 and IFN gamma was also frequently increased but not as consistently. The enhanced IFN gamma secretion associated with unchanged IL-4 secretion suggests that AvTK treatment results in a predominantly Th1-mediated antitumor immune response.
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PMID:Characterization of the antitumor immune response generated by treatment of murine tumors with recombinant adenoviruses expressing HSVtk, IL-2, IL-6 or B7-1. 947 56

Adenovirus-mediated gene transfer into the brain is associated with significant inflammation and activation of anti-vector and anti-transgene immune responses that curtail the gene delivery of adenoviruses and therapeutic efficacy. Elucidating the molecular mediators of inflammatory and immune responses to adenoviruses injected into the brain should allow us to inhibit their inflammatory actions, thereby reducing vector clearance and enhance adenoviral-mediated gene transfer into the CNS. Cytokines are primary mediators of the immune response and are released during inflammation. Here we report for the first time that injection of replication-deficient adenovirus vectors into the cerebral ventricles of rats causes a rapid increase in body temperature. This fever response precedes any vector-encoded transgene expression and occurs with vectors encoding no transgene, as well as with vectors encoding a therapeutic transgene i.e., HSV1-thymidine kinase. No fever is detected after infection of the striatum, an important brain target in studies on neurodegeneration. After infection of the brain ventricles, CSF levels of immunoreactive tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta increase significantly (up to 300-fold). In the hypothalamus, the locus of thermoregulation in the brain, only IL-1beta and IL-6 are significantly elevated. A neutralizing TNF-alpha antibody has no effect on adenovirus-induced fever. However, pretreatment with either the IL-1 receptor antagonist or the cyclooxygenase inhibitor flurbiprofen completely abolishes adenovirus-induced fever, suggesting that IL-1 and prostaglandins are direct mediators of this response. These results are the first to demonstrate that IL-1, but not TNF-alpha, is the main mediator of a very early inflammatory response to adenovirus in the brain.
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PMID:Interleukin-1 mediates a rapid inflammatory response after injection of adenoviral vectors into the brain. 995 27

Estrogens are important for bone homeostasis and are classified as antiresorptive agents. One of the mechanisms for this effect is the inhibition of cytokine-induced bone resorption, which is mediated in part through an interaction between the estrogen receptor (ER) and nuclear factor (NF)-kappaB in osteoblasts. We present evidence that bone-resorbing cytokines that activate NF-kappaB conversely inhibit ligand-dependent ER activity in the conditionally immortalized human osteoblast cell line, HOB-03-CE6. Treatment of HOB-03-CE6 cells with 17beta-estradiol (17beta-E2) up-regulated reporter gene activity [ERE-thymidine kinase (tk)-luciferase] 3- to 5-fold in a dose-dependent manner (EC50 = 1.0 pM). However, cotreatment of the cells with 17beta-E2 and increasing concentrations of either tumor necrosis factor-alpha (TNF alpha), interleukin-1alpha (IL-1alpha), or IL-1beta completely suppressed ERE-tk-luciferase activity in a dose-dependent manner (IC50 = 0.05-5.0 pM). On the other hand, treatment of the cells with growth factors either up-regulated or had no effect on ERE-tk-luciferase expression. Neither TNF alpha, IL-1alpha, nor IL-1beta treatment affected basal reporter gene activity in the cells, and the TNF alpha effect was reversed by a neutralizing antibody to the cytokine. TNF alpha treatment also suppressed ligand-dependent ER activity in MCF-7 human breast cancer cells, but not in Chinese hamster ovary cells that overexpressed human ER alpha, even though both cell lines responded to the cytokine as measured by the up-regulation of NFkappaB-tk-luciferase activity. TNF alpha treatment did not affect the steady state levels of either ER alpha or ER beta messenger RNA expression by the HOB-03-CE6 cells, nor did it reduce [125I]17beta-E2 binding. Moreover, TNF alpha treatment only weakly inhibited ligand-dependent glucocorticoid receptor activity in the HOB-03-CE6 cells. Bone-resorbing cytokines, which do not signal through the NF-kappaB pathway, did not suppress ERE-tk-luciferase activity in HOB-03-CE6 cells. Treatment of the cells with 17beta-E2 partially suppressed the activation of NF-kappaB by TNF alpha, but did not block cytokine-induced IL-6 secretion. Finally, cotreatment of HOB-03-CE6 cells with an antisense oligonucleotide to NF-kappaB p50 partially reversed the suppression of ERE-tk-luciferase activity by TNF alpha. In summary, these data provide evidence for a potent feedback inhibition of estrogen action in human osteoblasts that is at least partly mediated by the activation of NF-kappaB.
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PMID:Suppression of ligand-dependent estrogen receptor activity by bone-resorbing cytokines in human osteoblasts. 1034 28


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