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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antigen triggering of the T-cell receptor results in an accumulation of activated GTP-bound p21ras protein. To assess the role of ras protein in T-cell activation we have cotransfected the murine thymoma line EL4 with a construct capable of expressing a constitutively active, oncogenic form of Ha-ras and a reporter construct containing the human interleukin-2 promoter fused upstream of the bacterial gene for chloramphenicol acetyltransferase. We show that the ras oncoprotein contributes to interleukin-2 promoter activation. Its pattern of synergism with a calcium ionophore or the lymphokine interleukin-1 indicates that it replaces a signal mediated by protein kinase C. Interleukin-2 promoter activity in the presence of ras oncoprotein was inhibited by H7, a potent inhibitor of protein kinase C, but not by HA1004, an inhibitor of cyclic nucleotide-dependent kinase, suggesting that protein kinase C mediates the ras effect. In addition, we show that in these cells, expression of activated ras results in activation of a synthetic promoter containing several copies of an NF kappa B binding site.
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PMID:Interleukin-2 promoter activation in T-cells expressing activated Ha-ras. 153 20

We have used an interleukin-2 (IL-2) promoter-CAT fusion gene to study activation of IL-2 gene expression by IL-1, phytohemagglutinin (PHA), phorbol myristate acetate (PMA), and calcium ionophore in the murine thymoma line EL4 and the human lymphoma line Jurkat. The two cell lines respond differently to combinations of these stimuli. IL-1 in combination with suboptimal concentration of PMA induced chloramphenicol acetyltransferase (CAT) activity in EL4. In Jurkat cells, IL-1 failed to synergize with PMA or PHA. Cotransfection with the IL-2/CAT gene and a construct capable of expressing murine T-cell type IL-1 receptors converted Jurkat cells to IL-1 responsiveness. IL-1 in combination with PHA but not with PMA resulted in induction of CAT activity in these cells. Induction of IL-2/CAT activity by all stimuli in both cell lines was blocked by the presence of EGTA in the culture medium. EGTA did not inhibit IL-1/PMA activation of an SV40 early promoter-CAT fusion gene in either EL4 or Jurkat cells; therefore, calcium was not required for IL-1 or PMA signal transduction. Jurkat cells were shown to differ from EL4 in their requirement for calcium mobilization. Two different calcium-dependent pathways of gene activation were distinguished, both of which were blocked by the immunosuppressive drug cyclosporin A.
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PMID:Cyclosporin A blocks calcium-dependent pathways of gene activation. 165 71

Interleukin-1 (IL-1) is known to synergize with phorbol esters in the induction of interleukin-2 (IL-2) expression in T-lymphoid leukemia cells and proliferation of mouse thymocytes. We used a plasmid construct containing the bacterial gene for chloramphenicol acetyltransferase under the control of the human IL-2 promoter to study the nature of this synergism in the murine thymoma cell line EL4. Although IL-1 induction of the IL-2 promoter in these cells required costimulus with phorbol myristate acetate, the signal induced by IL-1 was qualitatively different. We provide evidence to support the hypothesis that the phorbol ester signal is mediated by protein kinase C, and we show that the IL-1 signal is not. That IL-1 and phorbol myristate acetate represent different stimuli was shown by their response to protein kinase C inhibitors, capacity to synergize with increased intracellular free calcium, and requirement for protein synthesis. In addition we show that pretreatment with IL-1 can prime EL4 cells to subsequent activation by concentrations of phorbol esters not normally sufficient to induce IL-2 expression. Pretreated cells remained primed for at least 40 h after removal of the IL-1. Neither phorbol myristate acetate nor a calcium ionophore was capable of preactivating EL4 cells.
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PMID:A role for protein kinase C activity in interleukin-1 (IL-1) induction of IL-2 gene expression but not in IL-1 signal transduction. 169 59

Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region.
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PMID:Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells. 191 35

Recombinant human interleukin-1 (IL-1) alpha and beta were found to activate a latent cytosolic form of the transcription factor NF kappa B in rat C6 glioma. IL-1 beta was 10 times more potent than IL-1 alpha for this activity and both were inhibited by the IL-1 receptor antagonist. The activation was detectable from 20 min and remained sustained for up to 24 h. The electrophoretic mobility of the activated complex was shown to be different from that of the corresponding complexes in another IL-1-responsive cell line, the murine thymoma line EL4.NOB-1. C6 cells, when transiently transfected with five NF kappa B consensus sequence repeats linked to the reporter gene chloramphenicol acetyltransferase (CAT), demonstrated increased CAT activity in response to IL-1 beta treatment. The activation of NF kappa B in glial cells may thus represent an early step in IL-1 signalling in brain and is likely to have consequences for IL-1-induced gene expression in these cells.
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PMID:Interleukin-1 activates transcription factor NF kappa B in glial cells. 837 49

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.
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PMID:Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes. 865 60

As an approach to understanding the molecular mechanism(s) of thymic gene expression mediated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we investigated the effect of TCDD on expression of prostaglandin G/H synthase-2 (PGHS-2) in rat thymocytes by reverse transcription-polymerase chain reaction. Incubation of thymocytes with increasing doses of TCDD resulted in inhibition of PGHS-2 gene expression in a concentration-dependent manner, with an IC50 of 10 nM. In contrast, TCDD had no appreciable effect on expression of glyceraldehyde phosphate dehydrogenase. Because the xenobiotic-responsive element is conserved in the PGHS-2 promoter from several animal species, it seems likely that inhibition of PGHS-2 expression by TCDD may occur at the level of transcription. To test this hypothesis in cultured thymocytes, we characterized the Ah receptor in the thymoma cell line WEHI 7.1. Reverse transcription-polymerase chain reaction experiments indicated that TCDD inhibited PGHS-2 expression in this cell line. Sucrose density gradient centrifugation experiments indicated that WEHI 7.1 cytosol exhibited 9 to 10S ligand-binding activity characteristic of the Ah receptor. The viability of WEHI 7.1 cells incubated with TCDD was comparable to that of control cells, whereas dexamethasone induced toxicity in a concentration-dependent manner. Transient transfection experiments using PGHS-2 promoter fragments ligated into a chloramphenicol acetyltransferase reporter plasmid suggested that TCDD inhibits PGHS-2 transcription, and deletion of the xenobiotic-responsive element failed to exhibit this repression. These results demonstrate that TCDD is a potent inhibitor of PGHS-2 gene expression, and they represent the first mechanistic evidence for TCDD-dependent inhibition of transcription in thymocytes.
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PMID:2,3,7,8-tetrachlorodibenzo-p-dioxin modulates expression of the prostaglandin G/H synthase-2 gene in rat thymocytes. 896 84

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