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)

A construct comprising three tandemly repeated copies of the kappaB element from the interleukin-8 gene linked to chloramphenicol acetyltransferase (CAT) (3xNF-kappaBCAT) was transcriptionally activated in normal human FS-4 fibroblasts by co-transfection with expression vectors for NF-kappaB p50, p65, or p52. Unexpectedly, a significant activation of 3xNF-kappaBCAT was also seen upon its co-transfection with the expression vector for CCAAT box enhancer binding protein alpha (C/EBP-alpha) (but not C/EBP-beta or C/EBP-delta). Stimulation by C/EBP-alpha required some other factor(s) present in FS-4 cells because no transcriptional activation of 3xNF-kappaBCAT was seen after co-transfection with C/EBP-alpha in F9 mouse embryonic carcinoma cells, known to be deficient in several transcription factors. To determine whether transcriptional activation was the result of interaction with one of the major NF-kappaB proteins, we co-transfected C/EBP-alpha with NF-kappaB p50, p65, p50 + p65, or p52 into F9 or FS-4 cells. No cooperative interaction was seen; in fact, C/EBP- alpha reduced p65-stimulated transcription, especially in F9 cells. Electrophoretic mobility shift assay with a kappaB probe revealed that the addition of recombinant C/EBP-alpha protein to nuclear extracts from untreated FS-4 cells resulted in the appearance of four bands. Only one of these bands was supershifted by antibody to p50, whereas antibodies to p65 or other NF-kappaB proteins had no effect. Our findings show that C/EBP-alpha may cause activation of some kappaB element-containing genes lacking C/EBP binding sites.
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PMID:CCAAT box enhancer binding protein alpha (C/EBP-alpha) stimulates kappaB element-mediated transcription in transfected cells. 862 20

Both the aging of animals and the senescence of cultured cells involve an altered pattern of gene expression, suggesting changes in transcription factor regulation. We studied age-related changes in transcription factors nuclear factor (NF)-kappa B, activator protein factor-1 (AP-1) and Sp-1 by using electrophoretic mobility shift binding assays; we also analysed changes in the protein components of NF-kappa B complex with Western blot assays. Nuclear and cytoplasmic extracts were prepared from heart, liver, kidney and brain of young adult and old NMRI mice and Wistar rats as well as from presenescent, senescent and simian virus 40-immortalized human WI-38 fibroblasts. Aging of both mice and rats induced a strong and consistent increase in the nuclear binding activity of NF-kappa B factor in all tissues studied, whereas those of AP-1 and Sp-1 decreased, e.g. in liver. Protein levels of p50, p52 and p65 components of the NF-kappa B complex did not show any age-associated changes in the cytoplasmic fraction but in the nuclear fraction the level of p52 strongly increased in heart and liver during aging. The protein levels of inhibitory I kappa B-alpha and Bcl-3 components were not affected by aging in any of the tissues studied. Replicative cellular senescence of human WI-38 fibroblasts induced a strong decrease in nuclear NF-kappa B, AP-1 and Sp-1 binding activities. Protein levels of p50 and p52 components of NF-kappa B complex were decreased in the nuclear fraction of senescent WI-38 fibroblasts but in the cytoplasm of senescent fibroblasts the level of p65 protein was increased. Cellular senescence also slightly decreased the protein levels of I kappa B-alpha and Bcl-3. Transfection assays with NF-kappa B-enhancer-driven chloramphenicol acetyltransferase reporter gene showed a significant down-regulation of NF-kappa B promoter activity in senescent WI-38 fibroblasts. Results suggest that the aging process might be regulated differently in tissues and cultured fibroblasts, perhaps reflecting differences between mitotic and post-mitotic cells. In tissues, aging seems to involve specific changes in the regulation of NF-kappa B components and perhaps also changes in the DNA-binding affinities of the NF-kappa B complex.
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PMID:Changes associated with aging and replicative senescence in the regulation of transcription factor nuclear factor-kappa B. 880 53

Bradykinin (BK), a pluripotent nonameric peptide, is known for its proinflammatory functions in both tissue injury and allergic inflammation of the airway mucosa and submucosa. To understand the mechanisms by which BK serves as an inflammatory mediator, the human lung fibroblast cell line WI-38 was stimulated with BK and the expression of IL-1beta gene was examined. BK at nanomolar concentrations induced a marked increase in immunoreactive IL-1beta, detectable within 2 h in both secreted and cell-associated forms. BK-induced IL-1beta synthesis was inhibited by a B2-type BK receptor antagonist and by treatment of the cells with pertussis toxin, indicating the involvement of a BK receptor that couples to the G(i)/G(o) class of heterotrimeric G proteins. Whereas cycloheximide and actinomycin D both inhibited BK-induced IL-1beta synthesis, results from Northern blot and nuclear run-on assays suggested that BK acted primarily at the transcription level which led to the accumulation of IL-1beta message in stimulated cells. Gel mobility shift assays were used with nuclear extracts from stimulated WI-38 cells to examine the transcription mechanism for BK-induced IL-1beta expression. A DNA binding activity specific for the decameric kappaB enhancer was detected and was found to contain the p50 and p65 subunits of the NF-kappaB/rel protein family. BK-induced NF-kappaB activation correlated with IL-1beta message upregulation with respect to agonist concentration, time course, sensitivity to bacterial toxins, and blockade by the B2 receptor antagonist. After BK stimulation, a significant increase in the activity of chloramphenicol acetyltransferase was observed in WI-38 cells transfected with a reporter plasmid bearing the kappaB enhancers from the IL-1beta gene. Deletion of the kappaB enhancer sequence significantly reduced BK-induced chloramphenicol acetyltransferase activity. These findings suggests a novel function of BK in the activation of NF-kappaB and the induction of cytokine gene expression.
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PMID:Bradykinin stimulates NF-kappaB activation and interleukin 1beta gene expression in cultured human fibroblasts. 890 23

Nuclear factor kappaB (NF-kappaB) is an important cellular regulator of human immunodeficiency virus (HIV) gene expression. In T cells, N-acetyl-L-cysteine (NAC) inhibits the induction of NF-kappaB and transcription of HIV-1. However, NAC up-regulates HIV-1 replication in monocyte-derived macrophages (MDM). In this study we demonstrate that NAC treatment of MDM transfected with a chloramphenicol acetyltransferase (CAT) construct under transcriptional control of the HIV-1 long terminal repeat resulted in an up-regulation of CAT activity. Furthermore, MDM transfected with a HIV-1-NF-kappaB-CAT construct also produced increased CAT activity after NAC treatment. In addition, electrophoretic mobility shift assays revealed that nuclei of NAC-treated MDM contained increased binding activity to wild-type, but not mutant, kappaB oligonucleotides. Components of the binding activity were identified with antibodies as the NF-kappaB subunits p50 and p65. These data indicate that NAC-induced enhancement of HIV-1 replication in MDM is regulated at the level of viral gene expression and mediated by NF-kappaB.
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PMID:N-acetyl-L-cysteine-induced up-regulation of HIV-1 gene expression in monocyte-derived macrophages correlates with increased NF-kappaB DNA binding activity. 900 May 34

The antitumor antibiotic mitomycin C is activated by several bioreductive enzymes, including DT-diaphorase. In HT29 cells, mitomycin C treatment results in the induction of DT-diaphorase as reflected in elevated steady state DT-diaphorase mRNA levels. An increase in the transcriptional rate was demonstrated by nuclear run-on assay. To investigate the molecular basis of the change in transcriptional activity caused by mitomycin C treatment, electrophoretic mobility shift assays were used to demonstrate the induction of nuclear factor binding to elements in the 5' flanking region of the DT-diaphorase gene. Treatment of HT29 cells with mitomycin C resulted in the dose-dependent induction of binding activity directed to the activator protein-1 (AP-1) binding element with a time course similar to that of mRNA elevation. Supershift assays using specific antibodies to Jun and Fos demonstrated the participation of both proteins in the binding activities generated. A binding activity for the nuclear factor-kappaB (NF-kappaB) site was induced with a similar time course. Both competitor and supershift experiments indicated that a heterodimer of the NF-kappaB proteins p50 and p65 was contained in the bound complex. To further investigate the functional consequences of such binding, we transfected HT29 cells with a plasmid containing 3 kb of the DT-diaphorase 5' region upstream of a reporter gene, chloramphenicol acetyltransferase. Treatment with mitomycin C resulted in a 5.5-fold increase in the expression of a chloramphenicol acetyltransferase construct containing 3 kb of DT-diaphorase promoter sequence. Using a series of deletion mutations based on this full-length construct, we found that two regions of the DT-diaphorase promoter region, positions -346 to -588 (containing the AP-1 element) and positions -785 to -890 (containing the NF-kappaB element) are required for the full expression of the mitomycin C response. The specific involvement of these binding elements was confirmed using mutational analysis. The results demonstrate that mutation of either element alone or of both diminishes the response, indicating an additive interaction between the elements at a minimum. However, inducibility characterizes a promoter fragment as small as 78 base-pairs from the transcription start site. Treatment of cells with mitomycin C induced binding to a 38-base-pair region (-40 to -78) devoid of known transcription factor binding elements. These data suggest that mitomycin C induces the overexpression of DT-diaphorase through a mechanism involving both the AP-1 and NF-kappaB response elements and that inducibility depends on a novel factor binding element.
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PMID:Involvement of activator protein-1 and nuclear factor-kappaB transcription factors in the control of the DT-diaphorase expression induced by mitomycin C treatment. 905 97

The "long pentraxins" are an emerging family of genes that have conserved in their carboxy-terminal halves a pentraxin domain homologous to the prototypical acute phase protein pentraxins (C-reactive protein and serum amyloid P component) and acquired novel amino-terminal domains. In this report, a genomic fragment of 1371 nucleotides from the human "long pentraxin" gene PTX3 is characterized as a promoter on tumor necrosis factor-alpha (TNFalpha) and interleukin (IL)-1beta exposure in transfected 8387 human fibroblasts by chloramphenicol acetyltransferase and RNase protection assays. In the same cells, the PTX3 promoter does not respond to IL-6 stimulation. Furthermore, IL-1beta and TNFalpha responsiveness is not seen in the Hep 3B hepatoma cell line. The minimal promoter contains one NF-kappaB element which is shown to be necessary for induction and able to bind p50 homodimers and p65 heterodimers but not c-Rel. Mutants in this site lose the ability to bind NF-kappaB proteins and to respond to TNFalpha and IL-1beta in functional assays. Sp1- and AP-1 binding sites lying in proximity to the NF-kappaB site do not seem to play a major role for cytokine responsiveness. Finally, cotransfection experiments with expression vectors validate that the natural promoter contains a functional NF-kappaB site.
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PMID:Characterization of the promoter for the human long pentraxin PTX3. Role of NF-kappaB in tumor necrosis factor-alpha and interleukin-1beta regulation. 907 34

Drug-induced cytotoxicity or apoptosis may be influenced by the expression of the p53 tumor suppressor gene and by the specific oncogene expressed, which may dictate the threshold at which a cytotoxic response may by induced. The objective of the study was to elucidate how DNA-damaging agents with different mechanisms of action were sensitized in the context of expression of the Pax3/FKHR fusion protein, a transformation event unique to alveolar rhabdomyosarcomas (ARMSs), and wild-type p53 (wtp53). A wtp53 cDNA was subcloned into the pGRE5-2/EBV vector with dexamethasone-inducible overexpression and transfected into Rh30 ARMS cells that express Pax3/FKHR and a mutant p53 phenotype. Following dexamethasone induction of wtp53 overexpression in a derived clone (Cl.#27), growth was slowed, and cells accumulated in G1. Functional wtp53 activity was demonstrated by selective transactivation of p50-2, a wtp53 chloramphenicol acetyltransferase reporter construct, and by up-regulated expression of endogenous p21Waf1. Data demonstrated p53-dependent sensitization (> or = 4-fold) to bleomycin, actinomycin D, and 5-fluorouracil and considerably less p53-dependence (< or = 2-fold) for doxorubicin, topotecan, etoposide, and cisplatin in Cl.#27 compared to an equivalent clone containing the pGRE5-EBV vector alone (VC#3). Data demonstrate that ARMS cells show a selective sensitization to DNA-damaging agents when wtp53 is overexpressed. The cytotoxic activity of agents that are not potentiated substantially must, therefore, depend upon p53-independent factors that relate to the mechanism of drug action.
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PMID:Selective sensitization to DNA-damaging agents in a human rhabdomyosarcoma cell line with inducible wild-type p53 overexpression. 951 63

The LYT-10 gene was initially cloned by virtue of its disruption by the translocation breakpoint in some t(10;14) lymphoid neoplasms. LYT-10 is now known to encode a component of the NF-kappaB family of transcriptional activators and has therefore also been designated NFkappaB2. Activation of NF-kappaB is generally associated with its transfer to the nucleus and is followed by a rapid increase in expression of its target genes, which include cytokines such as interleukin-6 (IL-6). IL-6 can also be induced by other transcription factors such as NF-IL6. We studied the interaction of IL-1 and these transcription factors in two renal cell carcinoma cell lines (ACHN and Caki-1). These lines produce high levels of IL-6, show endogenous chloramphenicol acetyltransferase activity for the IL-6 promoter, and have high basal levels of transcripts encoding the NF-kappaB components Lyt-10, p50, and p65 as well as the NF-IL6 transcription factor. IL-1alpha and IL-1beta markedly increased steady-state levels of LYT-10 (NFkappaB2) transcripts and nuclear Lyt-10 protein in both cell lines. Levels of the NFkappaB1 (p50-encoding), p65, and NF-IL6 transcripts also increased after IL-1 exposure. These changes were accompanied by a 20-fold or greater increase in levels of IL-6 messenger ribonucleic acid (mRNA) and protein. Our observations suggest that the mechanism by which IL-1alpha or IL-1beta induces IL-6 may be mediated through increases in LYT-10 mRNA and protein levels as well as increases in expression of other transcription factors (NFkappaB1, p65, and NF-IL6), in addition to the known ability of IL-1 to post-translationally activate NF-kappaB.
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PMID:Interleukin-1 increases expression of the LYT-10 (NFkappaB2) proto-oncogene/transcription factor in renal cell carcinoma lines. 952 51

Expression of the inducible nitric oxide synthase (iNOS) gene in rat mesangial cells is differentially triggered by IL-1beta and cAMP predominantly at the transcriptional level. The 5'-flanking region of the rat iNOS gene contains several binding sites for transcription factors potentially involved in cytokine and cAMP signaling such as nuclear factor-kappaB/Rel, CCAAT/enhancer-binding protein (C/EBP), and cyclic AMP-responsive element-binding protein/ATF. We tested promoter activities of serial and site-directed deletion mutants of iNOS-chloramphenicol acetyltransferase reporter genes after transient transfection and stimulation of mesangial cells. A region between bp -277 and -111 bearing a CCAAT/enhancer-binding protein-response element was found to be critical for cAMP-mediated gene induction but dispensable for IL-1beta inducibility. Moreover, a minimal promoter ranging from the transcriptional start site up to -111 containing a kappaB site is sufficient to confer IL-1beta-mediated iNOS promoter activation. Consistent with these findings, an electrophoretic mobility shift assay shows the appearance of an IL-1beta-inducible nuclear factor-kappaB p50/p65 heterodimeric complex. Using probes containing C/EBP-binding sites from the iNOS gene revealed further binding of different complexes, all of which were strongly inducible by cAMP and to a lower extent also by IL-1beta. Abs against cyclic AMP-responsive element-binding protein, C/EBPbeta, and C/EBPdelta were able to partially supershift single complexes, suggesting the participation of these transcription factors in the regulation of iNOS gene expression by cAMP and IL-1beta. Finally, we show that both cAMP and IL-1beta strongly induce steady-state levels of C/EBPbeta and C/EBPdelta mRNA levels. These data demonstrate that IL-1beta and cAMP use distinct as well as partially overlapping sets of transcriptional activators to modulate iNOS gene expression in rat mesangial cells.
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PMID:Molecular mechanisms of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat mesangial cells. 959 Feb 44

The role of ceramide as a second messenger in tumor necrosis factor (TNF)-mediated signal transduction has been much debated. It is supported by recent reports describing an expanding number of potential targets for this lipid, but is opposed by those describing how ceramide is not necessary for many TNF-mediated cellular events. In this paper, we directly compare the effects of the cell-permeable ceramide analogue, N-acetylsphingosine (C2-ceramide), with TNF, on NFkappaB function, a transcription factor whose activation is central to many TNF-mediated effects. We describe how C2-ceramide failed to drive kappaB-linked chloramphenicol acetyltransferase gene expression in either HL60 promyelocytic or Jurkat T lymphoma cells. Furthermore, it had no effect on TNF-mediated transcription of this reporter gene. However, electrophoretic mobility shift analysis following cell stimulation with this ceramide analogue revealed a dose-responsive activation of NFkappaB, which was not apparent following cell treatment with the inactive dihydro form. Activated complexes from treated cells were shown to contain predominantly the p50 subunit, in contrast to complexes from TNF-treated cells, where both p50 and p65/RelA subunits were present. The specific activation of p50 homodimeric complexes by C2-ceramide, which are known to lack trans-activating activity, was strongly suggested from these data. Further investigations revealed that C2-ceramide had only a marginal effect on IkappaBalpha degradation but strongly promoted the processing of p105 to its p50 product as revealed by immunoblot analysis. The increase in p50 arising from the processing of its p105 precursor was further established from p105/p50 ratios obtained by scanning densitometric analysis of bands from immunoblots. TNF, on the other hand, stimulated both IkappaBalpha degradation and p105 processing, in accordance with previous findings. Furthermore, the effect of TNF on NFkappaB activation was rapid, whereas C2-ceramide required an optimal treatment time of 1 h. Interestingly, TNF was found to increase ceramide in cells but only after a 1-h contact time. Our data therefore suggest that ceramide promotes the activation of NFkappaB complexes that lack transactivating activity by enhanced processing of p105.
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PMID:Ceramide activates NFkappaB by inducing the processing of p105. 962 36


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