Silencing of the DEK gene induces apoptosis and senescence in CaSki cervical carcinoma cells via the up-regulation of NF-κB p65

2012 ◽  
Vol 32 (3) ◽  
pp. 323-332 ◽  
Author(s):  
Kuiran Liu ◽  
Tianda Feng ◽  
Jie Liu ◽  
Ming Zhong ◽  
Shulan Zhang
Keyword(s):  
Cell Cycle ◽  
Nuclear Translocation ◽  
Annexin V ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Cell Senescence ◽  
Stable Cell Line ◽  
Reverse Transcription Pcr ◽  
Dna Binding Activity ◽  
Nuclear Extracts

The human DEK proto-oncogene has been found to play an important role in autoimmune disease, viral infection and human carcinogenesis. Although it is transcriptionally up-regulated in cervical cancer, its intracellular function and regulation is still unexplored. In the present study, DEK and IκBα [inhibitor of NF-κB (nuclear factor κB) α] shRNAs (short hairpin RNAs) were constructed and transfected into CaSki cells using Lipofectamine™. The stable cell line CaSki–DEK was obtained after G418 selection. CaSki–IκB cells were observed at 48 h after psiRNA-IκB transfection. The inhibitory efficiency of shRNAs were detected by RT (reverse transcription)–PCR and Western blot analysis. The proliferation activity of cells were measured using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, cell apoptosis was measured using an Annexin V/PI (propidium iodide) kit, the cell cycle was analysed by flow cytometry and cell senescence was detected using senescence β-galactosidase staining. The intracellular expression of NF-κB p65 protein was studied by cytochemistry. The expression levels of NF-κB p65, p50, c-Rel, IκBα and phospho-IκBα protein were analysed by immunoblotting in whole-cell lysates, cytosolic fractions and nuclear extracts. The protein expression and activity of p38 and JNK (c-Jun N-terminal kinase) were also assayed. In addition, the NF-κB p65 DNA-binding activity was measured by ELISA. Following the silencing of DEK and IκBα, cell proliferation was inhibited, apoptosis was increased, the cell cycle was blocked in the G0/G1-phase with a corresponding decrease in the G2/M-phase, and cell senescence was induced. All of these effects may be related to the up-regulation of NF-κB p65 expression and its nuclear translocation.

scholarly journals Human Herpesvirus 6B Induces Cell Cycle Arrest Concomitant with p53 Phosphorylation and Accumulation in T Cells

2005 ◽  
Vol 79 (3) ◽  
pp. 1961-1965 ◽  
Author(s):  
Bodil Øster ◽  
Bettina Bundgaard ◽  
Per Höllsberg
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Kinase Inhibitor ◽  
Human Herpesvirus ◽  
Annexin V ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
M Phase ◽  
Infected Cells ◽  
T Cell Lines

ABSTRACT We studied the interactions between human herpesvirus 6B (HHV-6B) and its host cell. Productive infections of T-cell lines led to G1/S- and G2/M-phase arrest in the cell cycle concomitant with an increased level and enhanced DNA-binding activity of p53. More than 70% of HHV-6B-infected cells did not bind annexin V, indicating that the majority of cells were not undergoing apoptosis. HHV-6B infection induced Ser20 and Ser15 phosphorylation on p53, and the latter was inhibited by caffeine, an ataxia telangiectasia mutated kinase inhibitor. Thus, a productive HHV-6B infection suppresses T-cell proliferation concomitant with the phosphorylation and accumulation of p53.

scholarly journals Interleukin-1β up-regulates RGS4 through the canonical IKK2/IκBα/NF-κB pathway in rabbit colonic smooth muscle

2008 ◽  
Vol 412 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Wenhui Hu ◽  
Fang Li ◽  
Sunila Mahavadi ◽  
Karnam S. Murthy
Keyword(s):  
Smooth Muscle ◽  
Target Gene ◽  
Nuclear Translocation ◽  
Specific Inhibitor ◽  
Muscle Cells ◽  
Nuclear Factor Κb ◽  
Inhibitory Effect ◽  
Binding Activity ◽  
Interleukin 1Β ◽  
Dna Binding Activity

Initial Ca2+-dependent contraction of the intestinal smooth muscle mediated by Gq-coupled receptors is attenuated by RGS4 (regulator of G-protein signalling 4). Treatment of colonic muscle cells with IL-1β (interleukin-1β) inhibits acetylcholine-stimulated initial contraction through increasing the expression of RGS4. NF-κB (nuclear factor κB) signalling is the dominant pathway activated by IL-1β. In the present study we show that RGS4 is a new target gene regulated by IL-1β/NF-κB signalling. Exposure of cultured rabbit colonic muscle cells to IL-1β induced a rapid increase in RGS4 mRNA expression, which was abolished by pretreatment with a transcription inhibitor, actinomycin D, implying a transcription-dependent mechanism. Existence of the canonical IKK2 [IκB (inhibitor of NF-κB) kinase 2]/IκBα pathway of NF-κB activation induced by IL-1β in rabbit colonic muscle cells was validated with multiple approaches, including the induction of reporter luciferase activity and endogenous NF-κB-target gene expression, NF-κB-DNA binding activity, p65 nuclear translocation, IκBα degradation and the phosphorylation of IKK2 at Ser177/181 and p65 at Ser536. RGS4 up-regulation by IL-1β was blocked by selective inhibitors of IKK2, IκBα or NF-κB activation, by effective siRNA (small interfering RNA) of IKK2, and in cells expressing either the kinase-inactive IKK2 mutant (K44A) or the phosphorylation-deficient IκBα mutant (S32A/S36A). An IKK2-specific inhibitor or effective siRNA prevented IL-1β-induced inhibition of acetylcholine-stimulated PLC-β (phopsholipase C-β) activation. These results suggest that the canonical IKK2/IκBα pathway of NF-κB activation mediates the up-regulation of RGS4 expression in response to IL-1β and contributes to the inhibitory effect of IL-1β on acetylcholine-stimulated PLC-β-dependent initial contraction in rabbit colonic smooth muscle.

UV stimulation induces nuclear factor κB (NF-κB) DNA-binding activity but not transcriptional activation

2001 ◽  
Vol 29 (6) ◽  
pp. 688-691 ◽  
Author(s):  
K. J. Campbell ◽  
N. R. Chapman ◽  
N. D. Perkins
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Cellular Response ◽  
Uv Light ◽  
Binding Protein ◽  
Nuclear Factor Κb ◽  
Camp Response Element Binding ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Dna Binding Activity

The cellular response to DNA-damaging agents is partly mediated by DNA-binding transcription factors such as p53 and nuclear factor κB (NF-κB). Typically NF-κB activation is associated with resistance to apoptosis. Following stimulation with UV light however, NF-κB activation has been shown to be required for programmed cell death. To study this effect further and to determine the relationship between NF-κB and p53 function, we have examined the effect of UV light on U2OS cells. UV stimulation resulted in the activation of NF-κB DNA-binding and the induction of p53. Surprisingly, and in contrast with tumour necrosis factor α stimulation, this UV-induced NF-κB was transcriptionally inert. These observations suggest a model in which the NF-κB switch from an anti-apoptotic to a pro-apoptotic role within the cell results from modulation of its ability to stimulate gene expression, possibly as a result of the ability of p53 to sequester transcriptional co-activator proteins such as p300/CREB (cAMP-response-element-binding protein)-binding protein.

scholarly journals 16K prolactin induces NF-kappaB activation in pulmonary fibroblasts

2002 ◽  
Vol 175 (3) ◽  
pp. R13-R18 ◽  
Author(s):  
Y Macotela ◽  
C Mendoza ◽  
AM Corbacho ◽  
G Cosio ◽  
JP Eiserich ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nuclear Translocation ◽  
Signal Transduction Pathway ◽  
Binding Activity ◽  
Inos Expression ◽  
Pulmonary Fibroblasts ◽  
Amino Terminal ◽  
Dna Binding Activity ◽  
Nf Kappab ◽  
Oxide Synthase

The amino-terminal 16 kDa fragment of prolactin (16K PRL) promotes the expression of the inducible isoform of nitric oxide synthase (iNOS) accompanied by the production of nitric oxide (NO) by rat pulmonary fibroblasts. The present study was designed to elucidate whether the mechanism by which 16K PRL promotes iNOS expression involves the activation of nuclear factor-kappa B (NF-kappaB), a key transcription factor for iNOS induction. 16K PRL stimulated DNA-binding activity of NF-kappaB in pulmonary fibroblasts as demonstrated by gel shift assays. Likewise, fluorescence immunocytochemistry showed that 16K PRL promotes nuclear translocation of the p65 subunit of NF-kappaB. Finally, treatment with 16K PRL induced the degradation of the NF-kappaB inhibitor kappaB-beta (IkappaB-beta), and such degradation was prevented by blocking IkappaB-beta phosphorylation. Altogether, these results show that 16K PRL activates NF-kappaB nuclear translocation via the phosphorylation and degradation of IkappaB-beta. These findings are consistent with NF-kappaB being part of the signal transduction pathway activated by 16K PRL to induce iNOS expression.

p65 controls NF-κB activity by regulating cellular localization of IκBβ

2011 ◽  
Vol 434 (2) ◽  
pp. 253-263 ◽  
Author(s):  
Taras Valovka ◽  
Michael O. Hottiger
Keyword(s):  
Dna Binding ◽  
Nuclear Export ◽  
Cellular Localization ◽  
Nuclear Export Signal ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Cellular Processes ◽  
Dna Binding Activity ◽  
Localization Signal ◽  
Export Signal

NF-κB (nuclear factor κB) controls diverse cellular processes and is frequently misregulated in chronic immune diseases or cancer. The activity of NF-κB is regulated by IκB (inhibitory κB) proteins which control nuclear–cytoplasmic shuttling and DNA binding of NF-κB. In the present paper, we describe a novel role for p65 as a critical regulator of the cellular localization and functions of NF-κB and its inhibitor IκBβ. In genetically modified p65−/− cells, the localization of ectopic p65 is not solely regulated by IκBα, but is largely dependent on the NLS (nuclear localization signal) and the NES (nuclear export signal) of p65. Furthermore, unlike IκBα, IκBβ does not contribute to the nuclear export of p65. In fact, the cellular localization and degradation of IκBβ is controlled by the p65-specific NLS and NES. The results of our present study also reveal that, in addition to stimulus-induced redistribution of NF-κB, changes in the constitutive localization of p65 and IκBβ specifically modulate activation of inflammatory genes. This is a consequence of differences in the DNA-binding activity and signal responsiveness between the nuclear and cytoplasmic NF-κB–IκBβ complexes. Taken together, the findings of the present study indicate that the p65 subunit controls transcriptional competence of NF-κB by regulating the NF-κB/IκBβ pathway.

Brief hypoxic stress suppresses postbacteremic NF-κB activation and TNF-α bioactivity in perfused liver

2000 ◽  
Vol 279 (1) ◽  
pp. R99-R108 ◽  
Author(s):  
Laura L. Loftis ◽  
Cheryl A. Johanns ◽  
Andrew J. Lechner ◽  
George M. Matuschak
Keyword(s):  
Gene Expression ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Mobility Shift ◽  
Gram Negative ◽  
Nuclear Extracts ◽  
Tnf Α ◽  
Cellular Hypoxia ◽  
Electrophoretic Mobility Shift Assays ◽  
Rat Livers

Reductions in hepatic O2 delivery are common early after gram-negative bacteremic sepsis owing to cardiopulmonary dysfunction and derangements in sinusoidal perfusion. Although gram-negative endotoxin and cellular hypoxia independently enhance activation of nuclear factor-κB (NF-κB) via generation of reactive O2 species (ROS), the combination of these stimuli downregulates hepatic TNF-α gene expression. Here we tested the hypothesis that hypoxic suppression of postbacteremic TNF-α gene expression is transcriptionally mediated by reduced activation of NF-κB. Buffer-perfused rat livers ( n = 52) were studied over 180 min after intraportal infection at t = 0 with 109 live Escherichia coli (EC), serotype O55:B5, or 0.9% NaCl controls under normoxic conditions, compared with 0.5 h of constant-flow hypoxia (Po 2 ∼41 ± 7 Torr) beginning at t = 30 min, followed by 120 min of reoxygenation. In parallel studies, tissue was obtained at peak hypoxia ( t = 60 min). To determine the role of xanthine oxidase (XO)-induced ROS in modulating NF-κB activity after hypoxia/reoxygenation (H/R), livers were pretreated with the XO inhibitor allopurinol, with results confirmed in organs of tungstate-fed animals. Electrophoretic mobility shift assays were performed on nuclear extracts of whole liver lysates using32P-labeled oligonucleotides specific for NF-κB. Compared with normoxic EC controls, hypoxia reduced postbacteremic NF-κB nuclear translocation and TNF-α bioactivity, independent of reoxygenation, tissue levels of reduced glutathione, or posthypoxic O2 consumption. XO inhibition reversed the hypoxic suppression of NF-κB nuclear translocation and ameliorated decreases in cell-associated TNF-α. Thus decreases in hepatic O2delivery reduce postbacteremic nuclear translocation of NF-κB and hepatic TNF-α biosynthesis by signaling mechanisms involving low-level generation of XO-mediated ROS.

scholarly journals Mild Hypothermia Inhibits Nuclear Factor-κB Translocation in Experimental Stroke

2003 ◽  
Vol 23 (5) ◽  
pp. 589-598 ◽  
Author(s):  
Hyung Soo Han ◽  
Murat Karabiyikoglu ◽  
Stephen Kelly ◽  
Raymond A. Sobel ◽  
Midori A. Yenari
Keyword(s):  
Target Genes ◽  
Nuclear Translocation ◽  
Mild Hypothermia ◽  
Brain Temperature ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Cerebral Injury ◽  
Experimental Stroke ◽  
Nuclear Factor ◽  
Iκb Kinase

Nuclear factor-κB (NFκB) is a transcription factor that is activated after cerebral ischemia. NFκB activation leads to the expression of many inflammatory genes involved in the pathogenesis of stroke. The authors previously showed that mild hypothermia is protective even when cooling begins 2 h after stroke onset. In the present study, they examined the influence of hypothermia on NFκB activation. Rats underwent 2 h of transient middle cerebral artery occlusion. Brains were cooled to 33°C immediately after or 2 h after occlusion, and maintained for 2 h. After normothermic ischemia (brain temperature at 38°C), NFκB cytoplasmic expression, nuclear translocation, and binding activity were observed as early as 2 h in the ischemic hemisphere and persisted at 24 h. Hypothermia decreased NFκB translocation and binding activity but did not alter overall expression. Hypothermia also affected the levels of NFκB regulatory proteins by suppressing phosphorylation of NFκB's inhibitory protein (IκB-α) and IκB kinase (IKK-γ) and decreasing IKK activity, but did not alter overall IKK levels. Hypothermia suppressed the expression of two NFκB target genes: inducible nitric oxide synthase and TNF-α. These data suggest that the protective effect of hypothermia on cerebral injury is, in part, related to NFκB inhibition due to decreased activity of IKK.

scholarly journals Methylation of STAT6 Modulates STAT6 Phosphorylation, Nuclear Translocation, and DNA-Binding Activity

2004 ◽  
Vol 172 (11) ◽  
pp. 6744-6750 ◽  
Author(s):  
Weiguo Chen ◽  
Michael O. Daines ◽  
Gurjit K. Khurana Hershey
Keyword(s):  
Dna Binding ◽  
Nuclear Translocation ◽  
Binding Activity ◽  
Dna Binding Activity

scholarly journals Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription.

1992 ◽  
Vol 116 (1) ◽  
pp. 31-42 ◽  
Author(s):  
S J Busch ◽  
G A Martin ◽  
R L Barnhart ◽  
M Mano ◽  
A D Cardin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Enhanced Recovery ◽  
Present Report ◽  
Binding Activity ◽  
Heparin Binding ◽  
Dna Binding Activity ◽  
Cycle Arrest ◽  
Specific Mrnas ◽  
F9 Teratocarcinoma

Heparin blocks the phorbol ester-induced progression of nontransformed cells through the G0/G1 phase (Wright, T.C., L.A. Pukac, J.J. Castellot, M.J. Karnovsky, R.A. Levine, H.-Y. Kim-Park, and J. Campisi. 1989. Proc. Natl. Acad. Sci. USA. 86: 3199-3203) or G1 to S phase (Reilly, C. F., M. S. Kindy, K. E. Brown, R. D. Rosenberg, and G. E Sonenshein. 1989. J. Biol. Chem. 264:6990-6995) of the cell cycle. Cell cycle arrest was associated with decreased levels of stage-specific mRNAs suggesting transcriptional regulation of cell growth. In the present report, we show that heparin selectively repressed TPA-inducible AP-1-mediated gene expression. Heparin-induced trans-repression was observed in primary vascular smooth muscle cells, as well as in the transformed HeLa cell line and in nondifferentiated F9 teratocarcinoma cells. Inhibition of AP-1-mediated trans-activation occurred with heparin and pentosan polysulfate but not with chondroitin sulfate A or C. Heparin-binding peptides or heparitinase I addition to nuclear lysates of heparin-treated cells allowed enhanced recovery of endogenous AP-1-specific DNA binding activity. We propose a model in which nuclear glycosaminoglycans play a trans-regulatory role in altering the patterns of inducible gene expression.

Regulatory Proteins Bind at the Polymorphic NFSE Element of CYP3A4.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4453-4453
Author(s):  
Tal David-Kalish ◽  
Deborah Rund ◽  
Elad Malik ◽  
Sara Bar Cohen
Keyword(s):  
Dna Binding ◽  
Cell Lines ◽  
Blast Crisis ◽  
Binding Activity ◽  
Dna Probe ◽  
Luciferase Expression ◽  
Cytochrome P450 Enzyme ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Nuclear Extracts

Abstract CYP3A4 is the most abundant cytochrome P450 enzyme in the liver and is involved in the metabolism of most clinically used drugs. An A to G substitution in the nifedipine responsive element (NFSE) in the promoter of this gene has been found to be associated with a lower incidence of pediatric therapy-related leukemia (Felix, Proc Natl Acad Sci USA95:13176, 1998) and adult therapy-related leukemia (Rund et al, Leukemia, accepted for publication). To study the effect of this polymorphism on gene expression in hematopoietic cells, we constructed reporter plasmids with the luciferase gene (in pGL3E) under control of the CYP3A4 promoter, using both the polymorphic and normal sequences. These plasmids were transfected into several cell lines of hematopoietic origin and luciferase was quantitated. We used KG1a (myeloid leukemia), K562 (CML blast crisis), and as controls, MelA1, a melanoma line and HepG2, a hepatoma line. Experiments were repeated at least three times for each cell line. The results consistently demonstrated 20–30% lower luciferase activity (in KG1a and K562 respectively) using the polymorphic sequence as compared to the normal sequence while the MelA1 and HepG2 lines showed the opposite effect, a 25% higher luciferase expression with the variant sequence. The results for HepG2 were in agreement with those reported by Rebbeck (Environmental and Molecular Mutagenesis49:299, 2003). To identify the factors binding at NFSE which may influence expression, electrophoretic mobility shift assays were performed using nuclear extracts of both cell lines (K562, KG1a, and HL60) and patient leukemia cells with a DNA probe representing the normal and polymorphic sequences. A gel shift was demonstrated, indicating binding of nuclear extracts to the region of the polymorphism. The database of transacting factors states complete homology of the polymorphic sequence of the NFSE region with the consensus binding site of HSF-1. We therefore performed a series of experiments to determine if HSF-1 is the protein binding at that site. HSF-1 is a multimeric transcription factor which binds to heat shock elements in many promoters which are rapidly transcribed following stress by increases in temperature. We found that recombinant HSF-1 did not bind to the DNA probe alone. However, nuclear extracts of cells which underwent stress by heating to 43°C for one hour (which is known to increase HSF-1 production) demonstrated increased binding to the probe representing the region of the polymorphism and Western blotting demonstrated more HSF-1 in these extracts. Using a Streptavidin-biotin system with a DNA fragment representing the NFSE region, we demonstrated that DNA binding activity to the probe was present in the elution fractions which contained HSF-1, as detected by ECL (enhanced chemoluminescence). Elution fractions which did not show DNA binding activity did not contain detectable HSF-1. We conclude that HSF-1 may be the protein which binds at the NFSE element of the CYP3A promoter but that it binds either as a multimer or as part of a complex of several proteins, which complicates its detection as a DNA binding protein.

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