Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Despite promoting growth in many cell types, epidermal growth factor (EGF) induces growth inhibition in a variety of cancer cells that overexpress its receptor. The cyclin-dependent kinase inhibitor p21(WAF1) is a central component of this pathway. We found in human MDA-468 breast cancer cells that EGF up-regulates p21(WAF1) mRNA and protein, through a combination of increased mRNA stability and transcription. The decay rate of a hybrid luciferase reporter full-length p21(WAF1) 3'-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of p21(WAF1) 3'-UTR constructs identified multiple cis-acting elements capable of reducing basal reporter activity. Short wavelength ultraviolet light induced reporter activity in constructs containing the 5' region of the p21(WAF1) 3'-UTR, whereas EGF induced reporter activity in constructs containing sequences 3' of the UVC-responsive region. These cis-elements bound multiple proteins from MDA-468 cells, including HuR and poly(C)-binding protein 1 (CP1). Immunoprecipitation studies confirmed that HuR and CP1 associate with p21(WAF1) mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21(WAF1) protein expression or growth inhibition. However, binding of HuR to its target 3'-UTR cis-element was regulated by UVC but not by EGF, suggesting that these stimuli modulate the stability of p21(WAF1) mRNA via different mechanisms. We conclude that EGF-induced p21(WAF1) protein expression is mediated largely by stabilization of p21(WAF1) mRNA elicited via multiple 3'-UTR cis-elements. Although HuR binds at least one of these elements, it does not appear to be a major modulator of p21(WAF1) expression or growth inhibition in this system. CP1 is a novel p21(WAF1) mRNA-binding protein that may function cooperatively with other mRNA-binding proteins to regulate p21(WAF1) mRNA stability.
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PMID:The 3'-untranslated region of p21WAF1 mRNA is a composite cis-acting sequence bound by RNA-binding proteins from breast cancer cells, including HuR and poly(C)-binding protein. 1243 87

We previously demonstrated that a differentiation inducing drug, vesnarinone induced the growth arrest and p21(waf1) gene expression in a human salivary gland cancer cell line, TYS. In the present study, we investigated the mechanism of the induction of p21(waf1) gene by vesnarinone in TYS cells. We constructed several reporter plasmids containing the p21(waf1) promoter, and attempted to identify vesnarinone-responsive elements in the p21(waf1) promoter. By the luciferase reporter assay, we identified the minimal vesnarinone-responsive element in the p21(waf1) promoter at -124 to -61 relative to the transcription start site. Moreover, we demonstrated by electrophoretic mobility shift assay that Sp1 and Sp3 transcription factors bound to the vesnarinone-responsive element. Furthermore, we found that vesnarinone induced the histone hyperacetylation in TYS cells. These results suggest that vesnarinone directly activates p21(waf1) promoter via the activation of Sp1 and Sp3 transcription factors and the histone hyperacetylation in TYS cells.
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PMID:Vesnarinone, a differentiation inducing drug, directly activates p21(waf1) gene promoter via Sp1 sites in a human salivary gland cancer cell line. 1243 98

Cellular CCAAT/enhancer binding protein alpha (C/EBPalpha) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at G(1)/S through stabilization of p21(CIP-1)/WAF1 and through transcriptional activation of the p21 promoter. The Epstein-Barr virus lytic-cycle transactivator protein ZTA is known to arrest the host cell cycle at G(1)/S via a p53-independent p21 pathway, but the detailed molecular mechanisms involved have not been defined. To further evaluate the role of ZTA in cell cycle arrest, we constructed a recombinant adenovirus vector expressing ZTA (Ad-ZTA), whose level of expression at a low multiplicity of infection in normal human diploid fibroblast (HF) cells was lower than or equal to the physiological level seen in Akata cells lytically induced by EBV (EBV-Akata cells). Fluorescence-activated cell sorting analysis of HF cells infected with Ad-ZTA confirmed that G(1)/S cell cycle arrest occurred in the majority of ZTA-positive cells, but not with an adenovirus vector expressing green fluorescent protein. Double-label immunofluorescence assays (IFA) performed with Ad-ZTA-infected HF cells revealed that only ZTA-positive cells induced the expression of both endogenous C/EBPalpha and p21 and blocked the progression into S phase, as detected by a lack of incorporation of bromodeoxyuridine. The stimulation of endogenous ZTA protein expression either through treatment with tetradecanoyl phorbol acetate in D98/HR1 cells or through B-cell receptor cross-linking with anti-immunoglobulin G antibody in EBV-Akata cells also coincided with the induction of both C/EBPalpha and p21 and their mRNAs, as assayed by Northern blot, Western blot, and IFA experiments. Mechanistically, the ZTA protein proved to directly interact with C/EBPalpha by coimmunoprecipitation in EBV-Akata cells and with DNA-bound C/EBPalpha in electrophoretic mobility shift assay experiments, and the in vitro interaction domain encompassed the basic leucine zipper domain of ZTA. ZTA also specifically protected C/EBPalpha from degradation in a protein stability assay with a non-EBV-induced Akata cell proteasome extract. Furthermore, both C/EBPalpha and ZTA were found to specifically associate with the C/EBPalpha promoter in chromatin immunoprecipitation assays, but the interaction with ZTA appeared to be mediated by C/EBPalpha because it was abolished by clearing with anti-C/EBPalpha antibody. ZTA did not bind to or activate the C/EBPalpha promoter directly but cooperatively enhanced the positive autoregulation of the C/EBPalpha promoter by cotransfected C/EBPalpha in transient luciferase reporter gene assays with Vero and HeLa cells as well as with DG75 B lymphocytes. Similarly, ZTA alone had little effect on the p21 promoter in transient reporter gene assays, but in the presence of cotransfected C/EBPalpha, ZTA enhanced the level of C/EBPalpha activation. This effect proved to require a previously unrecognized region in the proximal p21 promoter that contains three high-affinity C/EBPalpha binding sites. Finally, in C/EBPalpha-deficient mouse embryonic fibroblasts (MEF), Ad-ZTA was unable to induce either p21 or G(1) arrest, whereas it was able to induce both in wild-type MEF. Overall, we conclude that C/EBPalpha is essential for at least one pathway of ZTA-induced G(1) arrest during EBV lytic-cycle DNA replication and that this process involves a physical piggyback interaction between ZTA and C/EBPalpha leading to greatly enhanced C/EBPalpha and p21 levels through both transcriptional and posttranslational mechanisms.
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PMID:CCAAT/enhancer binding protein alpha interacts with ZTA and mediates ZTA-induced p21(CIP-1) accumulation and G(1) cell cycle arrest during the Epstein-Barr virus lytic cycle. 1250 63

Twenty-three nondiabetic volunteers were divided into three groups. In group A (n = 9), the glucose infusion was adjusted to maintain blood glucose at 5 mmol/l (euglycemic clamp). In group B (n = 9), the glucose infusion was adjusted to maintain blood glucose at 10 mmol/l (hyperglycemic clamp) over 2 h. Group C consisted of five volunteers who were studied as the control group. Peripheral blood mononuclear cells (PBMCs) were isolated before and at the end of a 2-h clamp. In group C, PBMCs were isolated before and after 2 h without performing a clamp. The euglycemic clamp as well as "no clamp" had no effects on all parameters studied. In contrast, a significant increase in carboxymethyllysine (CML) content and p21(ras) and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation was observed at the end of a 2-h hyperglycemic clamp. The nuclear factor (NF)-kappaB (but not Oct-1) binding activity increased significantly in the hyperglycemic clamp. Western blots confirmed NF-kappaB-p65-antigen translocation into the nucleus. IkappaBalpha did not change significantly in both groups. Hyperglycemia-mediated NF-kappaB activation and increase of CML content, p21(ras), and p42/44 MAPK phosphorylation was also seen in ex vivo-isolated PBMCs stimulated with 5 or 10 mmol/l glucose. Addition of insulin did not influence the results. Inhibition of activation of ras, MAPK, or protein kinase C blocked hyperglycemia-mediated NF-kappaB activation in ex vivo-isolated PBMCs stimulated with 10 mmol/l glucose. Similar data were obtained using an NF-kappaB-luciferase reporter plasmid. Therefore, we can conclude that an acute hyperglycemia-mediated mononuclear cell activation is dependent on activation of ras, p42/p44 MAPK phosphorylation, and subsequent NF-kappaB activation and results in transcriptional activity in PBMCs.
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PMID:Acute hyperglycemia causes intracellular formation of CML and activation of ras, p42/44 MAPK, and nuclear factor kappaB in PBMCs. 1260 1

DNA methylation in the promoter of certain genes is associated with transcriptional silencing. Methylation affects gene expression directly by interfering with transcription factor binding and/or indirectly by recruiting histone deacetylases through methyl-DNA-binding proteins. In this study, we demonstrate that the human lung cancer cell line H719 lacks p53-dependent and -independent p21(Cip1) expression. p53 response to treatment with gamma irradiation or etoposide is lost due to a mutation at codon 242 of p53 (C-->W). Treatment with depsipeptide, an inhibitor of histone deacetylase, was unable to induce p53-independent p21(Cip1) expression because the promoter of p21(Cip1) in these cells is hypermethylated. By analyzing luciferase activity of transfected p21(Cip1) promoter vectors, we demonstrate that depsipeptide functions on Sp1-binding sites to induce p21(Cip1) expression. We hypothesize that hypermethylation may interfere with Sp1/Sp3 binding. By using an electrophoretic mobility shift assay, we show that, although methylation within the consensus Sp1-binding site did not reduce Sp1/Sp3 binding, methylation outside of the consensus Sp1 element induced a significant decrease in Sp1/Sp3 binding. Depsipeptide induced p21(Cip1) expression was reconstituted when cells were pretreated with 5-aza-2'-deoxycytidine. Our data suggest, for the first time, that hypermethylation around the consensus Sp1-binding sites may directly reduce Sp1/Sp3 binding, therefore leading to a reduced p21(Cip1) expression in response to depsipeptide treatment.
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PMID:Methylation of adjacent CpG sites affects Sp1/Sp3 binding and activity in the p21(Cip1) promoter. 1277 51

LRP16 is a novel gene cloned from lymphocytic cells, and its function is not known. The expression level of LRP16 mRNA was up-regulated by estrogen in breast cancer MCF-7 cells based on the computed aided serial analysis of gene expression (SAGE) analysis. In this study, we investigate the effect of 17beta-estradiol (17beta-E(2)) on the expression of LRP16 mRNA and the effects of overexpression of LRP16 on the proliferation of cultured MCF-7 cells and the possible mechanisms involved. The expression level of LRP16 mRNA induced by 17beta-E(2) was determined by Northern blot analysis. LRP16 promoter-controlled luciferase expression vector (pGL3-S(0)) was co-transfected with various nuclear receptors, including estrogen receptor alpha and beta (ERalpha and ERbeta), glucocorticoid receptor alpha (GRalpha), androgen receptor (AR) and peroxisome-proliferator activated receptor gamma and alpha (PPARgamma and PPARgamma) into COS-7 cells, and the relative luciferase activity was measured using Dual-luciferase report assay systems. The effect of overexpression of LRP16 on MCF-7 proliferation was examined by the Trypan Blue exclusion method, and the cell cycle was analyzed by flow cytometry. The expression levels of cyclin E, p53 and p21(WAF1/CIP1) proteins were determined by Western blot analysis. The results showed (1) 17beta-E(2) induced a five- to eightfold increase in LRP16 mRNA levels in MCF-7 cells; (2) the relative luciferase activities in the COS-7 cells co-transfected by pGL3-S(0) and ERalpha or AR were 7.8-fold and 11-fold respectively of those in the control cells transfected by pGL3-S(0) alone; (3) overexpression of LRP16 stimulated MCF-7 cell proliferation, and the numbers of cells in the S-phase of the cell cycle in cells transfected with LRP16 increased about 10% compared with the control cells; and (4) cyclin E levels were much higher in cells with overexpression of LRP16 than in the control cells, while the expression levels of p53 and p21(WAF1/CIP1) were not different between the two groups of cells. From these results we concluded that estrogen up-regulates the expression level of LRP16 mRNA through activation of ERalpha and that overexpression of LRP16 promotes MCF-7 cell proliferation probably by increasing cyclin E.
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PMID:Up-regulation of LRP16 mRNA by 17beta-estradiol through activation of estrogen receptor alpha (ERalpha), but not ERbeta, and promotion of human breast cancer MCF-7 cell proliferation: a preliminary report. 1279 Jul 85

The p53 tumor suppressor is frequently inactivated in tumors by point mutations in the DNA-binding domain, resulting in loss of sequence-specific DNA binding and transcription function. We present evidence that ellipticine can restore the transactivation function of several transfected p53 mutants (175 H, 248W, 249S, 273 H, 281G), resulting in the induction of p53-responsive genes (p21(WAF1),MDM2) and activation of a p53-responsive luciferase reporter. Ellipticine also activates mutant p53 function in tumor cells expressing endogenous 194F, 233L, 241F, and 273C mutants. Treatment with ellipticine alters mutant p53 reactivity to conformation-sensitive Pab1620 and Pab240 antibodies and increases its sequence-specific DNA-binding activity in vivo. Finally, ellipticine activates mutant p53 and induces p21(WAF1) and MDM2 expression in nude mouse tumor xenografts. These results demonstrate that ellipticine can restore transcription function to mutant p53. This property may contribute to the selectivity of ellipticine-derived compounds against tumor cell lines expressing mutant p53.
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PMID:Rescue of mutant p53 transcription function by ellipticine. 1288 4

Lytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes G(1) cell cycle arrest mediated by the virus-encoded replication-associated protein (RAP) (or K8 protein), which induces high-level expression of the cellular C/EBPalpha and p21 proteins. Here we have examined the mechanism of this induction at both the transcriptional and posttranslational levels. RAP proved to bind very efficiently to both C/EBPalpha and p21 and stabilized them by up to 10-fold from proteasome-mediated degradation in vitro. Cross-linking revealed that RAP itself forms stable dimers and tetramers in solution and forms higher-order complexes but not heterodimers with C/EBPalpha. Cotransfection of RAP with C/EBPalpha cooperatively stimulated both the C/EBPalpha and p21 promoters in luciferase reporter gene assays. Only the basic/leucine zipper region of RAP was needed for interaction with and stabilization of C/EBPalpha, but both the N-terminal and C-terminal domains were required for transcriptional augmentation. In vitro-translated RAP interfered with DNA binding by C/EBPalpha in electrophonetic mobility shift assay (EMSA) experiments but did not itself bind to the target C/EBPalpha sites or form supershifted bands. However, in endogenous chromatin immunoprecipitation (ChIP) assays with tetradecanoyl phorbol acetate-induced PEL cells, RAP proved to specifically associate with the C/EBPalpha promoter in vivo, but only in a C/EBPalpha-dependent manner, implying an in vivo piggyback interaction with DNA-bound C/EBPalpha. Expression of exogenous RAP (Ad-RAP) caused G(1)/S cell cycle arrest in human dermal microvascular endothelial cells and also induced both the C/EBPalpha and p21 proteins, which formed punctate nuclear patterns that colocalized with RAP in PML nuclear bodies. In the presence of RAP, C/EBPalpha was also efficiently recruited into viral DNA replication compartments in both infected and cotransfected cells. In support of a direct role for this interaction in viral DNA replication, three C/EBPalpha binding sites were identified by in vitro EMSA experiments within a 220-bp core segment of the duplicated KSHV Ori-Lyt region, and although RAP did not bind to Ori-Lyt DNA directly in vitro, both endogenous RAP and C/EBPalpha were found to be associated with the Ori-Lyt region by ChIP assays in lytically induced PEL cells. Finally, we found that the KSHV lytic cycle could not be triggered by either synchronizing KSHV latently infected PEL cells in G(1) phase or inducing p21 in a C/EBPalpha-independent process.
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PMID:Cell cycle arrest by Kaposi's sarcoma-associated herpesvirus replication-associated protein is mediated at both the transcriptional and posttranslational levels by binding to CCAAT/enhancer-binding protein alpha and p21(CIP-1). 1288 7

Cancer is a disease in which the cell cycle is altered, and the elucidation of the mechanisms by which constituents of human fecal water influence the cell cycle can lead to noninvasive measurement of colon cancer risk. The purpose of the present study was to investigate the effect of human fecal water on HT-29 cell-cycle progression with sodium selenite as a reference for comparison. Both human fecal water (2.5-5.0%) and selenite (3-4 micro mol/L) significantly inhibited cell growth. Cell-cycle analysis revealed that human fecal water decreased the proportion of S + G2 phase cells and increased that of G1 phase cells. In contrast, selenite decreased G1 phase cells and increased proportions of S and G2 phase cells. Both 5% human fecal water and 4 micro mol/L selenite greatly increased the mRNA level of the cyclin-dependent kinase inhibitor gene p21(waf1). Interestingly, the mRNA levels of cyclin A and proliferating cell nuclear antigen (PCNA) were dramatically decreased by 69 and 62%, respectively, in HT-29 cells treated with fecal water but not selenite. In contrast, the mRNA level of DNA damage-inducible transcript 1, gadd45, was significantly increased by 2.28-fold in HT-29 cells treated with selenite but not fecal water. Furthermore, a PCNA gene promoter was cloned into a luciferase reporter construct and its activity was significantly reduced in a dose-dependent manner in cells treated with fecal water but not selenite. Collectively, these results suggest that human fecal water and selenite can differentially induce growth arrest genes, and that PCNA gene expression is uniquely and highly sensitive to human fecal water.
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PMID:Down-regulation of proliferating cell nuclear antigen gene expression occurs during cell cycle arrest induced by human fecal water in colonic HT-29 cells. 1288 58

Glucocorticoids and estrogens regulate a number of vital physiological processes. We developed a model breast cancer cell line, MCF-7 M, to examine potential mechanisms by which the ligand-bound estrogen receptor (ER) regulates glucocorticoid receptor (GR)-mediated transcription. MCF-7 cells, which endogenously express ERalpha, were stably transfected with mouse mammary tumor virus promoter-luciferase (MMTV-LUC) reporter and GR expression constructs. Our results demonstrate that treatment with estrogen agonists (17beta-estradiol [E2], diethylstilbestrol, genistein), but not antagonists (tamoxifen or raloxifene), for 48 h inhibits GR-mediated MMTV-LUC transcription and chromatin remodeling. Furthermore, estrogen agonists inhibit glucocorticoid induction of p21 mRNA and protein levels, suggesting that the repressive effect applies to other GR-regulated genes and proteins in MCF-7 cells. Importantly, GR transcriptional activity is compromised because treatment with estrogen agonists down regulates GR protein levels. The protein synthesis inhibitor cycloheximide and the proteasome inhibitor MG132 block E2-mediated decrease in GR protein levels, suggesting that estrogen agonists down regulate the GR via the proteasomal degradation pathway. In support of this, we demonstrate that E2-mediated GR degradation is coupled to an increase in p53 and its key regulator protein Mdm2 (murine double minute 2), an E3 ubiquitin ligase shown to target the GR for degradation. Using the chromatin immunoprecipitation assay, we demonstrate an E2-dependent recruitment of ERalpha to the Mdm2 promoter, suggesting a role of ER in the regulation of Mdm2 protein expression and hence the enhanced GR degradation in the presence of estrogen agonists. Our study shows that cross talk between the GR and ER involves multiple signaling pathways, indicative of the mechanistic diversity within steroid receptor-regulated transcription.
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PMID:Estrogen receptor-dependent proteasomal degradation of the glucocorticoid receptor is coupled to an increase in mdm2 protein expression. 1289 56


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