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)

Transcription of tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted protein that regulates the activities of the metalloproteinases, collagenase and stromelysin, is activated by serum growth factors. Transient transfection experiments have revealed several regions of cis-acting regulatory sequences involved in the response of the murine TIMP-1 gene to serum. One area is in the vicinity of the promoter, consisting of a non-consensus binding site (5'-TGAGTAA-3' at -59/-53) for transcription factor AP1 and an adjacent 24 bp region of dyad symmetry that contains a PEA3-binding site. A second is an upstream region (-1020 to -780) that acts as an enhancer when linked to a heterologous promoter, and contains a consensus AP1 binding site (at -803/ -797). Gel retardation assays revealed differences between nuclear factors in mouse C3H10T1/2 cells that bound to the TIMP(-59/ -53)AP1 site and a consensus collagenase TRE (TPA-response element). The TIMP(-59/ -53)AP1 site is a promiscuous motif that binds c-Fos/c-Jun AP1 translated in vitro and is an effective competitor for binding of nuclear AP1 factors to the consensus TRE, but in addition it binds factors that do not associate with the consensus TRE. The TIMP(-59/ -53)AP1 motif and the dyad symmetry region stimulated expression from a thymidine kinase promoter in an additive fashion, and competition experiments showed that excess copies of these factor binding sites reduced expression from a reporter plasmid driven by the TIMP-1 promoter. Our data show that binding sites for AP1 and PEA3 transcription factors are involved in the regulation of TIMP-1 transcription, which suggests that the coordinated induction of TIMP-1, collagenase and stromelysin may be achieved through the actions of a shared set of nuclear transcription factors. However, the properties of the TIMP-1(-59/ -53)AP1 motif likely reflect an additional type of transcriptional regulation restricted to TIMP-1.
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PMID:Involvement of AP1 and PEA3 binding sites in the regulation of murine tissue inhibitor of metalloproteinases-1 (TIMP-1) transcription. 142 Mar 63

3,5,3,'-Triiodothyroacetic acid (Triac) has been used in therapy of resistance to thyroid hormone on an empirical basis and appears beneficial in some studies. We observed that the T3 analogs, Triac and 3,5,3'-triiodothyropropionic acid (Triprop), have a higher affinity for the thyroid hormone receptor-beta 1 (TR beta 1) than does T3 (2.7- and 1.8-fold, respectively), whereas the affinities of the three compounds for TR alpha 1 are the same. To evaluate whether T3 analogs would have a differential effect on TR beta 1 and TR beta 1 mutants and thus be a specific treatment for patients with resistance to thyroid hormone, we examined the induction of the transcriptional activation of wild-type (wt) TR alpha 1, TR beta 1, and mutant TR beta 1s by T3, Triac, and Triprop. The dose response of transcriptional activation by T3 analogs was measured by transient cotransfections with TRs and a rat malic enzyme-TRE fused to thymidine kinase (TK)-chloramphenicol acetyltransferase (CAT) in COS-1 cells. For TR alpha 1 wt, induction of CAT activity by T3 and Triac occurred at the same concentration. For TR beta 1 wt, Triac and Triprop showed a higher maximal activity than T3 (Tripro > Triac > T3) and reached 50% induction at a lower concentration than T3 (Tripro < Triac < T3). Induction of CAT activity in five mutant TR beta 1s (kindreds Mh, Mc, CL, Mf, and GH) was also analyzed. Even high levels of T3 analogs could not restore CAT activity to that of TR beta 1wt for any mutant. A dominant negative effect was produced by Mh, Mc, and Mf. Mutants CL and GH had a mild dominant negative effect depending on T3 analog concentrations and TREs. Cotransfection studies were performed using a rat malic enzyme-TK-CAT reporter plasmid to analyze the effects of hormones at near-physiological concentrations of T3 and Triac. Triac had a significantly higher transcriptional activation than T3 in Mc, CL, and GH, suggesting that Triac would have a beneficial effect to different degrees for different mutant TR beta 1s. Using mutants Mc and GH, further studies were carried out using rat GH and double palindromic and inverted palindromic TREs in COS-1 cells. On each TRE, 10 nmol/L Triac induced higher transcriptional activation in TR beta 1wt, mutant TR beta 1s, and TR beta 1wt plus mutant TR beta 1s (1:1 ratio) than the same dose of T3.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Triiodothyroacetic acid has unique potential for therapy of resistance to thyroid hormone. 760 51

The current study demonstrates that T3-activated transcription of the NADPH:cytochrome P450 oxidoreductase (P450R) gene is dependent on the thyroid hormonal status of the animal, with both transcriptional and post-transcriptional pathways being important in regulating the cellular P450R mRNA level. The region required for transcriptional activation of the P450R gene by T3 has been identified. Nuclear run-on experiments demonstrated that the effects of T3 on P450R transcription are dependent on thyroid status, with a transcriptional enhancement obtained in T3-treated hypothyroid rat liver (1.8-fold increase) but not in T3-treated euthyroid animals. Transient cotransfection of P450R promoter/chloramphenicol acetyl transferase (CAT) constructs and the thyroid hormone receptor beta1 (TR beta1) expression plasmid into rat hepatoma H4IIE cells resulted in a 2.4-fold induction of promoter activity that was both T3 and TR beta1 dependent. Analysis of promoter deletion constructs identified a P450R-thyroid response region (P450R-TRE; bases, -564 to -536) containing three imperfect direct repeats of the thyroid response motif, AGGTCA. Mutational analysis further established that T3 induction was dependent only on the upstream direct repeat, having the sequence AGGTGAgctgAGGCCA. Footprint analysis showed that all three motifs were protected by proteins present in rat liver nuclear extracts, and a direct interaction between P450R-TRE and T3 receptors TR alpha1 and TR beta1 was demonstrated by gel-shift analysis. In vitro binding studies with P450R-TRE revealed the formation of heterodimeric complexes when TR alpha1 was coincubated with either the retinoic X receptor alpha or nuclear extract from rat liver, COS, or H4IIE cells. In addition, placement of the P450R-TRE upstream of the T3-nonresponsive heterologous thymidine kinase promoter resulted in a 2.7-fold transcriptional enhancement that was both T3 and TR beta1 dependent. Previous studies have demonstrated that T3 augments P450R mRNA levels approximately 20-30-fold and approximately 12-fold, respectively, in hypothyroid and euthyroid rats. Hence, for the hypothyroid state, transcriptional and post-transcriptional events contribute to the T3-induced mRNA increases; however, the marked increase in message level in T3-treated euthyroid animals depends primarily on post-transcriptional pathways.
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PMID:Thyroid regulation of NADPH:cytochrome P450 oxidoreductase: identification of a thyroid-responsive element in the 5'-flank of the oxidoreductase gene. 922 11

Previous studies in our laboratory show that triiodothyronine upregulates expression of the cerebellar Purkinje cell-specific gene Pcp-2 during the first 2 weeks of rat neonatal life. A specific thyroid hormone response element, the A1 TRE, mediates this regulation. The finding that the contiguous 68 bases (-267/ -199) of the Pcp-2 promoter 3' to the A1 TRE repressed T3 response in transactivation studies suggested that this sequence could play a role in preventing premature T3-dependent activation of Pcp-2 in the fetus. We now show that deletion of this region resulted in enhanced T3-dependent activation of the native Pcp-2 promoter. The sequence is not a generalized silencer since it does not alter basal activity of mouse mammary tumor virus (MMTV) or thymidine kinase (TK) promoters. Deletion and linker scanning studies indicate that the 5' 30 bases of the -267/ -199 region mediate most of the response silencing activity. The -267/ -199 region also attenuates T3-induced transactivation mediated by other TREs. Gel shift analysis reveals that nuclear proteins from fetal but not adult brains complex with the -267/ -199 region, supporting the hypothesis that this region binds proteins that suppress Pcp-2 expression early in brain development.
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PMID:Purkinje cell protein-2 cis-elements mediate repression of T3-dependent transcriptional activation. 925 66

Oxidant stress is associated with diminution of antioxidant molecules, such as alpha-tocopherol. Alpha-tocopherol specifically decreases, in a concentration dependent way, the proliferation of vascular smooth muscle cells. At the same concentrations (10-50 microM) it induces inhibition of protein kinase C (PKC) activity. The latter event is not due to a decrease in PKC level or to alpha-tocopherol binding to PKC, but it results from increase of protein phosphatase 2A1 activity. In vitro data, as well as at a cellular level, demonstrates that protein phosphatase 2A1 is activated, in its trimeric structure--but not as a dimer by alpha-tocopherol. This activation is followed by PKC-alpha dephosphorylation. The activation of protein phosphatase 2A1 and deactivation of PKC-alpha affect the AP1 transcription factor, resulting in a change in the composition and the binding of this factor to DNA. By transfecting smooth muscle cell with a construct containing three TRE (TPA responsive elements), the promoter thymidine kinase and the reporter gene chloramphenicol-acetyl-transferase a modulation of gene expression by alpha-tocopherol is observed. Beta-tocopherol does not cause any of the responses observed with alpha-tocopherol and R,R,R-alpha-tocopherol is twice as potent as all-rac-alpha-tocopherol. When added together, beta-tocopherol prevents the effects of alpha-tocopherol indicating that the mechanism involved is not related to the radical-scavenging properties of these two molecules, which are essentially equal. By differential display analysis it has been found that several genes of smooth muscle cells are differentially transcribed in the presence of alpha-tocopherol but not beta-tocopherol. In particular, the gene of alpha-tropomyosin shows a transient enhancement of transcription as a function of the cell cycle time. Alpha-tropomyosin translation is also increased by alpha-tocopherol and not by beta-tocopherol. Because no changes of mRNA stability can be observed in the presence of alpha-tocopherol, the data supports the conclusion of a transcriptional control exerted by alpha-tocopherol on alpha-tropomyosin. Generally, the data strongly suggests the existence of a ligand/receptor type of mechanism at the basis of alpha-tocopherol action. It is concluded that an oxidative stress-induced diminution of alpha-tocopherol in smooth muscle cell activates a reaction cascade leading to changes in gene expression and increase in cell proliferation by a non-antioxidant mechanism.
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PMID:Vitamin E mediated response of smooth muscle cell to oxidant stress. 1058 72

Colorectal cancer is the most common cancer in both men and women and the second most common cause of cancer-related deaths. Suicide gene-based therapy with suicide gene-transduced mesenchymal stem cells (MSCs) is a promising therapeutic strategy. A tetracycline-controlled Tet-On inducible system used to regulate gene expression may be a useful tool for gene-based therapies. The aim of this study was to develop therapeutic MSCs with a suicide gene that is induced by an artificial stimulus, to validate therapeutic gene expression, and to monitor the MSC therapy for colon cancer using optical molecular imaging. For our study, we designed the Tet-On system using a retroviral vector and developed a response plasmid RetroX-TRE (tetracycline response element) expressing a mutant form of herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Bone marrow-derived MSCs were transduced using a RetroX-Tet3G (Clontech, CA, USA) regulatory plasmid and RetroX-TRE-HSV1-sr39TK-eGFP-IRES-Fluc2, for a system with a Tet-On (MSC-Tet-TK/Fluc2 or MSC-Tet-TK) or without a Tet-On (MSC-TK/Fluc2 or MSC-TK) function. Suicide gene engineered MSCs were co-cultured with colon cancer cells (CT26/Rluc) in the presence of the prodrug ganciclovir (GCV) after stimulation with or without doxycycline (DOX). Treatment efficiency was monitored by assessing Rluc (CT26/Rluc) and Fluc (MSC-Tet-TK and MSC-TK) activity using optical imaging. The bystander effect of therapeutic MSCs was confirmed in CT26/Rluc cells after GCV treatment. Rluc activity in CT26/Rluc cells decreased significantly with GCV treatment of DOX(+) cells (p < 0.05 and 0.01) whereas no significant changes were observed in DOX(-) cells. In addition, Fluc activity in also decreased significantly with DOX(+) MSC-Tet-TK cells, but no signal was observed in DOX(-) cells. In addition, an MSC-TK bystander effect was also confirmed. We assessed therapy with this system in a colon cancer xenograft model (CT26/Rluc). We successfully transduced cells and developed a Tet-On system with the suicide gene HSV1-sr39TK. Our results confirmed the therapeutic efficiency of a suicide gene with the Tet-On system for colon cancer. In addition, our results provide an innovative therapeutic approach using the Tet-On system to eradicate tumors by administration of MSC-Tet-TK cells with DOX and GCV.
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PMID:Regulated Mesenchymal Stem Cells Mediated Colon Cancer Therapy Assessed by Reporter Gene Based Optical Imaging. 2958 88