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)

Induction of cytochrome (CYP) P4501A2 by such polycyclic aromatic hydrocarbons as 3-methylcholanthrene (3MC) can lead to the bioactivation of carcinogenic aromatic amines and heterocyclic amines. A 3MC response element was recently identified approximately 2.2 kb upstream of the transcription start site of the human CYP1A2 gene. Sequence analysis of this enhancer identified, in addition to a binding site for the aryl hydrocarbon receptor, two other sequences, referred to as 5'AP1 and 3'AP1, each with complete homology to the phorbol 12-O-tetradecanoate 13-acetate (TPA) response element consensus sequence. Nuclear extracts from TPA-treated HepG2 cells protected both the 5'AP1 and 3'AP1 sequences against digestion with DNase I. Gel mobility shift and supershift assays revealed that TPA treatment of HepG2 results in increased binding activity of the AP-1 proteins, c-Jun, JunD, and c-Fos, to both sites. We transiently expressed, in HepG2, either a fragment containing both the 5'AP1 and 3'AP1 sites (-2.3pT81Luc) or only the 3'AP1 site (-2.2pT81Luc) cloned into a plasmid containing the luciferase gene under transcriptional control of the thymidine kinase promoter. TPA treatment of cells transfected with -2.3pT81Luc resulted in an approximately threefold induction of luciferase activity over untreated control cells, while the -2.2pT81Luc construction containing only the 3'AP1 site displayed an approximately sixfold induction. These studies suggest that the human CYP1A2 gene may be regulated by tumor promoters in addition to polycyclic aromatic hydrocarbons.
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PMID:Induction of the human CYP1A2 enhancer by phorbol ester. 946 18

Human herpesvirus 8 (HHV-8) is a newly discovered virus closely associated with Kaposi's sarcoma and primary effusion lymphomas. When they occur in patients with AIDS, these B-cell lymphomas frequently harbor another human herpesvirus, Epstein-Barr virus (EBV). To determine the molecular mechanisms of the regulation of early gene expression by the immediate-early gene products of HHV-8 and to assess possible molecular interactions between HHV-8 and EBV, we studied the regulation of the HHV-8 thymidine kinase (TK) promoter in cell lines harboring either or both viruses. The constitutive chloramphenicol acetyltransferase (CAT) activity of the TK promoter was low in all six cell lines tested. A putative immediate-early gene product of HHV-8 ORF50, which is a homolog of EBV BRLF1, was cloned into an expression vector and tested for its transactivating capacity. In the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the CAT activity of the TK promoter was increased 7- to 720-fold by cotransfection with the ORF50 clone in EBV-producing cell lines (Ramos/AW, P3HR-1, and BC-1) but not in EBV-negative cell lines (BCBL-1 and Ramos), nor in the latently EBV-infected cell line Raji. The TK promoter contains three consensus SP1- and two AP1-binding sites. In electrophoretic mobility shift assays, the cellular factor SP1, but not AP1, was found to bind specifically to the TK promoter. To determine whether the increased CAT activity resulted from the interaction of SP1 with the ORF50 gene product, we introduced mutations into two SP1-binding sites. Both mutated SP1 sites had reduced SP1-binding activity and greatly decreased TK promoter responsiveness to ORF50 transactivation, suggesting that upregulation of TK promoter by ORF50 is SP1 dependent.
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PMID:Activation of human herpesvirus 8 (HHV-8) thymidine kinase (TK) TATAA-less promoter by HHV-8 ORF50 gene product is SP1 dependent. 977 32

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

Estrogens regulate the proliferation, cytoarchitectural, and invasive properties of estrogen receptor (ER)-containing breast cancer cells. To identify genes under direct regulation by estrogen in breast cancer cells, we have used representational difference analysis (RDA) of cDNAs. In this way, we have identified (cyto)keratin 19 (K19), a major component of cell intermediate filaments, as being under rapid and direct regulation by estrogen in MCF-7 cells. Stimulation by estradiol (E2) of K19 mRNA is rapid, with maximal increase at 3 h, and is not blocked by cycloheximide, suggesting that it is a primary response to the hormone. Increased accumulation of K19 protein is observable by 8 h after E2 and levels continue to increase at 24-48 h after E2 treatment. Suppression of E2-induced K19 gene expression by the antiestrogen ICI 182,780 suggests that ER mediates this regulation. Analysis of the human K19 chromosomal gene, by transient transfection assays employing reporter gene constructs with the 5' and 3' flanking regions and portions of the body of the K19 gene, has resulted in identification of a complex enhancer region in the first intron. This enhancer region consists of a near-consensus estrogen response element (K19 ERE, which differs by only 1 bp from the consensus ERE) and two ERE half sites, as well as two AP1-like sites. The results of transfections with either the K19 gene promoter or the heterologous thymidine kinase promoter and constructs containing mutated or deleted portions of the enhancer region show that the K19 ERE is responsible for the E2-dependent transactivation of the keratin 19 gene and for the synergism that is observed between E2 and TPA with both ER alpha and ER beta. These studies document ER regulation of the K19 gene, localize the estrogen responsive region, and suggest that up-regulation of keratin 19 gene expression by estrogen may contribute to the cytoskeletal and nuclear matrix reorganization, and increased metastatic potential of ER-containing breast cancer cells upon exposure to estrogens.
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PMID:Regulation of keratin 19 gene expression by estrogen in human breast cancer cells and identification of the estrogen responsive gene region. 1102 74

Colon cancer is the third and fourth most prevalent cancer among Iranian men and women, respectively. Suicide gene therapy is one of the alternative therapeutic modalities for cancer. The application of specific promoters for therapeutic genes should decrease the adverse effects of this modality. The combined aims of this study were to design a specific suicide gene therapy construct for colon cancer and study its effect in distinct representatives of transformed and nontransformed cells. The KRAS oncogene signaling pathway is one of the most important signaling pathways activated in colon cancer; therefore, we inserted the urokinase plasminogen activator receptor (uPAR; PLAUR gene) promoter as one of the upregulated promoters by this pathway upstream of a suicide gene (thymidine kinase [TK]) and a reporter gene (beta-galactosidase, beta-gal [LacZ]). This promoter is a natural combination of different motifs responsive to the RAS signaling pathway, such as the transcription factors AP1 (FOS/JUN), SP1, SP3, and AP2alpha, and nuclear factor kappa B (NFkappaB). The reporter plasmid under the control of the uPAR promoter (PUCUPARLacZ) had the ability to express beta-gal in colon cancer cells (human colon adenocarcinoma [SW480] and human colorectal carcinoma [HCT116] cell lines), while it could not express beta-gal in nontransformed human umbilical vein endothelial cells (HUVEC) and normal colon cells. After confirming the ability of pUCUPARTK (suicide plasmid) to express TK in SW480 and HCT116 cells by real-time PCR, cytotoxicity assays showed that pUCUPARTK decreased the viability of these cells in the presence of ganciclovir 20 and 40 microg/mL (and higher), respectively. Although M30 CytoDEATH antibody could not detect a significant rate of apoptosis induced by ganciclovir in pUCUPARTK-transfected HCT116 cells, the percentage of stained cells was marked in comparison with untreated cells. While this antibody could detect apoptosis in HCT116 cell line transfected with positive control plasmid, it could not detect apoptosis in SW480 cells transfected with the same positive control. This discrepancy could be attributed to the different mechanisms of TK/ganciclovir-induced apoptosis in tumor protein p53 (TP53)-expressing (HCT116) and -deficient (SW480) cells. Annexin-propidium iodide staining could detect apoptosis in treated, pUCUPARTK-transfected SW480 and HCT116 cells. This study showed that the uPAR promoter can be considered as a suitable candidate for specific suicide gene therapy of colon cancer and probably other cancers in which the RAS signaling pathway is involved in their carcinogenesis process.
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PMID:Selective suicide gene therapy of colon cancer exploiting the urokinase plasminogen activator receptor promoter. 2019 27


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