Incubation Of Nuclear Extracts Research Articles

Angiotensin II promotes poly(ADP-ribosyl)ation of c-Jun/c-Fos in cardiac fibroblasts

Although c-Jun/c-Fos (activator protein 1, AP1) contributes importantly to Ang II-induced cardiac fibrosis through induction of extracellular matrix protein over-expression in cardiac fibroblasts, the mechanism by which Ang II promotes c-Jun/c-Fos transactivation remains unclear. In this study, we demonstrated that c-Fos and c-Jun were poly(ADP-ribosyl)ated in cultured cardiac fibroblasts. Southwestern blot and EMSA assays showed that incubation of nuclear extracts with NAD + and active DNA increased the basal DNA binding activities of c-Jun (31.0 ± 1.0%, P < 0.01) and AP1 (14.2 ± 3.1%, P < 0.01); incubation of recombinant c-Fos or/and c-Jun with PARP-1, NAD + and active DNA increased the basal DNA binding activities of c-Jun (48.3 ± 4.2%, P < 0.01) and AP1 (21.2 ± 1.5%, P < 0.01). Treatment with Ang II promoted PARP-1 activation and enhanced poly(ADP-ribosyl)ation of c-Fos (14.1 + 1.1%, P < 0.01) and c-Jun (15.5 ± 5.6%, P < 0.01). Ang II also increased the basal DNA binding activities of c-Jun (13.5 ± 2.4%, P < 0.01) and AP1 (18.7 ± 3.5%, P < 0.01) in cultured cells. Inhibition of PARP-1 by PJ34 or siRNA effectively prevented Ang II-induced increases in the DNA binding of c-Jun and AP1, and decreased AP1-driven transcription (including collagen Iα1 and IIIα1, MMP-9 and TIMP-1). This study illustrated that c-Jun and c-Fos were poly(ADP-ribosyl)ated by PARP-1, and poly(ADP-ribosyl)ation enhanced the DNA binding of AP1. Ang II promoted poly(ADP-ribosyl)ation of c-Jun and c-Fos through activation of PARP-1 and, subsequently, enhanced AP1-driven transcription in cardiac fibroblasts.

Transcriptional Regulation of the SHP-1, a Candidate Tumor Suppressor Gene in Hematological Malignancies.

The SH2-Containing Protein-Tyrosine Phosphatase-1 (SHP-1) is a hematopoietic cell specific protein-tyrosine phosphatase and a candidate tumor suppressor. It has been shown that expression of SHP-1 is repressed in HTLV-1 transformed lymphocyte cell lines, Adult T-cell Leukemia and other hematological malignancies. However, the mechanisms that regulate SHP-1 expression remain unclear. We previously identified that the −120 to +157 of the SHP-1 P2 promoter (core promoter) exhibits similar levels of activity as the wild type promoter. Here we report the involvement of NF-kB and Oct-1 in the transcriptional regulation of the SHP-1 P2 promoter. To elucidate the mechanisms by which SHP-1 promoter is regulated, in vitro DNase I footprinting experiments were performed. Incubation of nuclear extracts from Jurkat cells with the SHP-1 P2 promoter probes revealed two DNase I protected regions between −100 and −90 and between −80 and −55 in the SHP-1 P2 promoter. Elk-1, Ik-1, NF-kB and Oct-1 were identified through database search as likely candidate factors binding to these regions. Electrophoretic Mobility Shift Assay (EMSA) was carried out using a double strand DNA probe that covers the previous DNase I footprinting area (−120 to −19) and Jurkat nuclear extract. Transcription factors NF-kB and Oct-1 were confirmed as binding to the SHP-1 P2 promoter through antibody supershift, probe competition, and/or site directed mutagenesis. Results from in vivo Chromatin Immunoprecipitation (ChIP) assay with anti-NF-kB and anti-Oct-1 antibody also strongly suggested that Oct-1 and NF-kB bound the SHP-1 P2 promoter in Jurkat cells. In transient transfection experiments using luciferase as a reporter, mutations at Oct-1 binding sites and NF-kB binding sites showed significant reduction in SHP-1 promoter activity compared to wild type core promoter, further indicating the involvement of these factors in the SHP-1 P2 promoter transcription regulation. In summary, this is the first report demonstrating that Oct-1 and NF-kB are involved in the regulation of SHP-1 P2 promoter. Modulation of these transcription factors could provide new targets for treatment of hematological malignancies.

Sp1 Is a Co-activator with Ets-1, and Net Is an Important Repressor of the Transcription of CTP:Phosphocholine Cytidylyltransferase α

Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT) encoded by the Pcyt1a and Pcyt1b genes. Previously, we identified an Ets-1-binding site located at -49/-47 in the promoter of Pcyt1a as an important transcriptional element involved in basal CTalpha transcription (Sugimoto, H., Sugimoto, S., Tatei, K., Obinata, H., Bakovic, M., Izumi, T., and Vance, D. E. (2003) J. Biol. Chem. 278, 19716-19722). In this study, we determined whether or not there were other important elements and binding proteins for basal CTalpha transcription in the Pcyt1a promoter, and if other Ets family proteins bind to the Ets-1-binding site. The results indicate the formation of a ternary complex with Ets-1 binding at -49/-47 and Sp1 binding at -58/-54 of the Pcyt1a promoter that is important for activating CTalpha transcription. When nuclear extracts of COS-7 cells expressing various Ets family repressors were incubated with DNA probes, binding of Net to the probes was observed. Net dose-dependently depressed the promoter-luciferase activity by 98%, even when co-expressed with Ets-1. RNA interference targeting Net caused an increase of endogenous CTalpha mRNA. After synchronizing the cell cycle in NIH3T3 cells, CTalpha mRNA increased at the S-M phase corresponding to an increase of Ets-1 mRNA and a decrease of Net mRNA. These results indicated that Net is an important endogenous repressor for CTalpha transcription.

E47 phosphorylation by p38 MAPK promotes MyoD/E47 association and muscle-specific gene transcription

Selective recognition of the E-box sequences on muscle gene promoters by heterodimers of myogenic basic helix-loop-helix (bHLH) transcription factors, such as MyoD, with the ubiquitous bHLH proteins E12 and E47 is a key event in skeletal myogenesis. However, homodimers of MyoD or E47 are unable of binding to and activating muscle chromatin targets, suggesting that formation of functional MyoD/E47 heterodimers is pivotal in controlling muscle transcription. Here we show that p38 MAPK, whose activity is essential for myogenesis, regulates MyoD/E47 heterodimerization. Phosphorylation of E47 at Ser140 by p38 induces MyoD/E47 association and activation of muscle-specific transcription, while the nonphosphorylatable E47 mutant Ser140Ala fails to heterodimerize with MyoD and displays impaired myogenic potential. Moreover, inhibition of p38 activity in myocytes precludes E47 phosphorylation at Ser140, which results in reduced MyoD/E47 heterodimerization and inefficient muscle differentiation, as a consequence of the impaired binding of the transcription factors to the E regulatory regions of muscle genes. These findings identify a novel pro-myogenic role of p38 in regulating the formation of functional MyoD/E47 heterodimers that are essential for myogenesis.

Modulation of activator protein 1/DNA binding activity by acoustic overstimulation in the guinea-pig cochlea

Changes in gene expression are part of the homeostatic machinery with which cells respond to external stimuli or assaults. The activity of the early response transcriptional factor activator protein-1 (AP-1) can be modulated by a variety of environmental stimuli including those that alter the cellular oxidation/reduction status. This study investigates the activation of AP-1/DNA binding in the guinea-pig cochlea in response to acoustic overstimulation which produces reactive oxygen species. Electrophoretic mobility shift assays revealed that binding of AP-1 to its radiolabeled oligonucleotide probe markedly changed in nuclear extracts of inner ear tissues following intense noise exposure (4 kHz octave band, 115 dB, 5 h). AP-1/DNA binding increased in the organ of Corti and the lateral wall tissues immediately after the exposure, returning to near-baseline levels 5 h later. At 15 h after noise, a second peak of binding activity occurred in the organ of Corti whereas stria vascularis showed a lesser but more sustained activity. Binding in nuclear extracts from the spiral ganglion did not change. Incubation of nuclear extracts with antibodies against Fos/Jun family proteins prior to a supershift assay showed Fra-2 as a major component of the AP-1 complex immediately after the noise exposure. In the organ of Corti, Fra-2 immunoreactivity was localized to the middle turn, i.e. the region which is most affected by the 4-kHz octave band exposure. The results suggest the modulation of gene expression via the activation of AP-1 as a consequence of noise trauma but also demonstrate differential responses in cochlear tissues.

Nuclear factor kappa B is involved in lipopolysaccharide-stimulated induction of interferon regulatory factor-1 and GAS/GAF DNA-binding in human umbilical vein endothelial cells.

1. In this study we examined the signalling events that regulate lipopolysaccharide (LPS)-stimulated induction of interferon regulatory factor (IRF)-1 in human umbilical vein endothelial cells (HUVECs). 2. LPS stimulated a time- and concentration-dependent increase in IRF-1 protein expression, an effect that was mimicked by the cytokine, tumour necrosis factor (TNF)-alpha. 3. LPS stimulated a rapid increase in nuclear factor kappa B (NFkappaB) DNA-binding activity. Pre-incubation with the NFkappaB pathway inhibitors, N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK) or pyrrolidine dithiocarbamate (PDTC), or infection with adenovirus encoding IkappaBalpha, blocked both IRF-1 induction and NFkappaB DNA-binding activity. 4. LPS and TNFalpha also stimulated a rapid activation of gamma interferon activation site/gamma interferon activation factor (GAS/GAF) DNA-binding in HUVECs. Preincubation with the Janus kinase (JAK)-2 inhibitor, AG490 blocked LPS-stimulated IRF-1 induction but did not affect GAS/GAF DNA-binding. 5. Preincubation with TLCK, PDTC or infection with IkappaBalpha adenovirus abolished LPS-stimulated GAS/GAF DNA-binding. 6. Incubation of nuclear extracts with antibodies to RelA/p50 supershifted GAS/GAF DNA-binding demonstrating the involvement of NFkappaB isoforms in the formation of the GAS/GAF complex. 7. These studies show that NFkappaB plays an important role in the regulation of IRF-1 induction in HUVECs. This is in part due to the interaction of NFkappaB isoforms with the GAS/GAF complex either directly or via an intermediate protein.

A novel protein complex distinct from mismatch repair binds thioguanylated DNA.

To elucidate molecular mechanism(s) of cellular response to mercaptopurine, a widely used antileukemic agent, we assessed mercaptopurine (MP) sensitivity in mismatch repair (MMR) proficient and MMR deficient human acute lymphoblastic leukemia (ALL) cells. Sensitivity to thiopurine cytotoxicity was not dependent on MMR (i.e., MutSalpha) competence among six cell lines tested. Using electrophoretic mobility shift assay analysis, we found that the incubation of nuclear extracts from ALL cells with synthetic 34-mer DNA duplexes containing deoxythioguanosine (G(S)) within either G(S).T or G(S).C pairs, resulted in formation of a DNA-protein complex distinct from the DNA-MutSalpha complex and unaffected by ATP. Isolation and sequence analysis of proteins involved in this DNA-protein complex identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a component. Western blot analysis of nuclear extracts from a panel of human lymphoblastic leukemia cell lines revealed markedly different basal levels of GAPDH in nuclei, which was significantly related to thiopurine sensitivity (p = 0.001). Confocal analysis revealed markedly different intracellular distribution of GAPDH between nucleus and cytosol in six human ALL cell lines. Redistribution of GAPDH from cytosol to nucleus was evident after MP treatment. These findings indicate that a new DNA-protein complex containing GAPDH and distinct from known MMR protein-DNA complexes binds directly to thioguanylated DNA, suggesting that this may act as a sensor of structural alterations in DNA and serve as an interface between these DNA modifications and apoptosis.

Cell-specific Transcription of Leukotriene C4Synthase Involves a Kruppel-like Transcription Factor and Sp1

Leukotriene C(4) synthase (LTC(4)S) is responsible for the biosynthesis of cysteinyl leukotrienes that participate in allergic and asthmatic inflammation. We analyzed 2.1 kilobases of the 5'-flanking region of the human LTC(4)S gene, which contains three DNase I hypersensitivity sites, for its transcriptional activity when fused to a promoterless and enhancerless luciferase gene. Deletion analysis revealed a nonspecific basal promoter region between nucleotides -122 and -56 upstream of the translation start site which contains a consensus Sp1 binding site and a putative initiator element (Inr) and cell-specific enhancer regions further upstream. A single mutation of either the Sp1 binding site between nucleotides -120 and -115 or the Inr (CAGAC) between nucleotides -66 and -62 reduced the expression of the reporter gene by approximately 60%, whereas double mutations decreased the expression by approximately 80%. The incubation of nuclear extracts from THP-1 and K562 cells with a (32)P-labeled oligonucleotide containing the Sp1 site or the Inr sequence gave gel-shifted complexes that were blocked by their respective cold oligonucleotides, and antisera specific for Sp1 and Sp3 provided supershifts for the former. Linker-scanning mutations of a cell-specific regulatory region revealed that mutations from nucleotides -165 to -125 reduced reporter activity. This region contains a tandem CACCC repeat (at nucleotides -149 to -145 and -139 to -135). An oligonucleotide containing the distal CACCC motif was gel shifted by THP-1 cell nuclear extract and was supershifted by antisera to Sp1 and Sp3. Cotransfection of an Sp1 expression plasmid into Drosophila SL2 cells with a -228 to -3 LTC(4)S reporter construct transactivated the reporter gene, whereas mutations at the CACCC repeat region reduced Sp1 transactivation by approximately 66%. Similarly, the Kruppel-like factor Zf9/CPBP (core promoter-binding protein) transactivated the -228 construct in COS cells but not its CACCC mutant construct. These findings indicate the involvement of Sp1 and an Inr in non-cell-specific regulation and a Kruppel-like transcription factor and Sp1 in the cell-specific regulation of the LTC(4)S gene. These are the first such analyses of a member of a newly recognized superfamily of membrane-associated proteins involved in eicosanoid and glutathione metabolism, which contains key proteins involved in the generation of both prostanoids and cysteinyl leukotrienes.

Differential Regulation of Macrophage Peroxisome Proliferator–Activated Receptor Expression by Glucose

Peroxisome proliferator-activated receptors (PPARs) are implicated in several metabolic disorders with altered glucose and lipid metabolism, including atherosclerosis and diabetes. In the present study, we evaluated the in vitro and ex vivo effects of high glucose concentrations on macrophage PPAR mRNA expression. Exposition of monocyte-derived macrophages isolated from healthy donors to a high glucose environment led to an increase in PPARalpha and PPARbeta mRNA expression. In contrast, this treatment significantly decreased human macrophage PPARgamma mRNA expression. Overexpression of PPARalpha and PPARbeta mRNA and inhibition of PPARgamma mRNA expression were also observed in monocyte-derived macrophages isolated from patients with type 2 diabetes. Because high glucose and PPARalpha agonists increase lipoprotein lipase (LPL) gene expression, the role of PPARalpha in the glucose-mediated upregulation of macrophage LPL gene expression was next evaluated. Incubation of murine J774 macrophages with high glucose concentrations increased the expression of PPARalpha at the mRNA and protein levels and enhanced nuclear protein binding to the peroxisome proliferator responsive element of the LPL promoter. Incubation of nuclear extracts in the presence of anti-PPARalpha and anti-PPARbeta antibodies decreased glucose-stimulated nuclear protein binding to the peroxisome proliferator responsive element. These results demonstrate that glucose is an important regulator of macrophage PPAR expression and suggest a role of PPARalpha and PPARbeta in the upregulation of macrophage LPL by glucose. Dysregulation of macrophage PPAR expression in type 2 diabetes may contribute, by altering arterial lipid metabolism and inflammatory response, to the accelerated atherosclerosis associated with diabetes.

Nuclear Factor of Activated T Cells (NFAT) as a Molecular Target for 1α,25-Dihydroxyvitamin D3-Mediated Effects

Abstract The molecular basis of the immunomodulatory properties of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) remains elusive. We demonstrate here that 1α,25(OH)2D3-mediated suppressive effects on the inducible expression of cytokine genes in human T cells may, in part, be due to diminished activity of the transcription factor NFAT. The vitamin D3 receptor (VDR) and its heterodimeric partner retinoid X receptor α (RXRα) specifically bound to the distal NFAT site in the human IL-2 promoter, and this binding was abolished by mutating unique regions in the NFAT oligonucleotide. In vitro inhibition of NFAT complex formation was noted when VDR-RXRα heterodimers were added to DNA binding reactions containing nuclear extracts from activated B or T cells, whereas in vitro NFκB complex formation was not significantly influenced. Furthermore, 1α,25(OH)2D3 treatment of activated T cells resulted in decreased formation of NFAT complexes detected upon incubation of nuclear extracts from these cells with 32P-labeled probe. Transient expression of both VDR and RXRα, but not of a single component, was capable of inhibiting expression of a NFAT-driven reporter gene in stimulated Jurkat cells in a ligand-dependent manner. These results suggest that NFAT plays a crucial role in 1α,25(OH)2D3-mediated immunosuppressive activity.

Role of estrogen receptor/Sp1 complexes in estrogen-induced heat shock protein 27 gene expression.

Treatment of MCF-7 human breast cancer cells with 10 nM 17 beta-estradiol (E2) resulted in a 2-fold induction of heat shock protein (Hsp) 27 mRNA levels, and this response persisted for up to 24 h. The 5'-promoter region of the gene was further investigated to identify genomic sequences associated with E2 responsiveness. An Sp1 and half-palindromic estrogen response element (ERE) separated by 10 nucleotides, GGGCGGG(N)10GGTCA, were identified at -105 to -84, and formation of the Sp1/estrogen receptor (ER) complex was investigated by in vitro assays using synthetic Hsp 27-[32P]Sp1/ERE oligonucleotides in a gel mobility shift assay and transient transfection studies using short (-108/-84) and long (-108/+23) 5'-promoter sequences linked to a thymidine kinase promoter and the bacterial chloramphenicol acetyl transferase (CAT) reporter gene (Hsp-CATs and Hsp-CATl, respectively). Incubation of nuclear extracts from MCF-7 cells with an Hsp 27-[32P]Sp1/ERE oligonucleotide results in formation of an Sp1/ER complex. The formation of this complex was inhibited by coincubation with unlabeled Sp1/ERE, ERE, and Sp1 oligonucleotides and by preincubation with ER or Sp1 antibodies (immunodepletion). In addition, the complex was supershifted by coincubation with ER antibodies. Mutation of either Sp1 or ERE sites also decreases formation of the retarded band. E2 induced CAT activity in MCF-7 cells transiently transfected with either Hsp-CATs or Hsp-CATl plasmids. It was also demonstrated that E2 did not significantly induce CAT activity in MCF-7 cells transiently transfected with Hsp-CATl-containing mutations in both the Sp1 and ERE sites. The results of this study demonstrate that an Sp1/ER complex is involved in E2-induced Hsp 27 gene expression.

Down-regulation of NF-kappa B protein levels in activated human lymphocytes by 1,25-dihydroxyvitamin D3.

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2)D3], a steroid hormone with immunomodulating properties, on nuclear factor kappa B (NF-kappa B) proteins was examined in in vitro activated normal human lymphocytes by Western blot analysis. Over a 72-hr period of activation, the expression of the 50-kDa NF-kappa B, p50, and its precursor, p105, was increased progressively. When cells were activated in the presence of 1,25(OH)2D3, the levels of the mature protein as well as its precursor were decreased. The effect of the hormone on the levels of p50 was demonstrable in the cytosolic and nuclear compartments; it required between 4 and 8 hr and was specific, as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were ineffective. Besides p50, 1,25(OH)2D3 decreased the levels of another NF-kappa B protein, namely c-rel. In addition, 1,25(OH)2D3 decreased the abundance of a specific DNA-protein complex formed upon incubation of nuclear extracts from activated lymphocytes with a labeled NF-kappa B DNA binding motif. Further, 1,25(OH)2D3 inhibited the transcriptional activity of NF-kappa B in Jurkat cells transiently transfected with a construct containing four tandem repeats of the NF-kappa B binding sequence of the immunoglobulin kappa light chain gene linked to the chloramphenicol acetyltransferase reporter gene. These observations demonstrate directly that there is de novo synthesis of NF-kappa B during human lymphocyte activation and suggest that this process is hormonally regulated.

Triggering of the Human Interleukin-6 Gene by Interferon-γ and Tumor Necrosis Factor-α in Monocytic Cells Involves Cooperation between Interferon Regulatory Factor-1, NFκB, and Sp1 Transcription Factors

We investigated the molecular basis of the synergistic induction by interferon-gamma (IFN-gamma)/tumor necrosis factor-alpha (TNF-alpha) of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and compared it with the basis of this induction by lipopolysaccharide (LPS). Functional studies with IL-6 promoter demonstrated that three regions are the targets of the IFN-gamma and/or TNF-alpha action, whereas only one of these regions seemed to be implicated in LPS activation. The three regions concerned are: 1) a region between -73 and -36, which is the minimal element inducible by LPS or TNF-alpha; 2) an element located between -181 and -73, which appeared to regulate the response to IFN-gamma and TNF-alpha negatively; and 3) a distal element upstream of -224, which was inducible by IFN-gamma alone. LPS signaling was found to involve NF kappa B activation by the p50/p65 heterodimers. Synergistic induction of the IL-6 gene by IFN-gamma and TNF-alpha, in monocytic cells, involved cooperation between the IRF-1 and NF kappa B p65 homodimers with concomitant removal of the negative effect of the retinoblastoma control element present in the IL-6 promoter. This removal occurred by activation of the constitutive Sp1 factor, whose increased binding activity and phosphorylation were mediated by IFN-gamma.

Phosphorylation of the Transcription Factor NFATp Inhibits Its DNA Binding Activity in Cyclosporin A-treated Human B and T Cells

Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phosphatase calcineurin, which in turn inhibits translocation of an NFAT component to the nucleus. Here we report that CsA treatment of Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp that is inhibited in DNA-binding and in its ability to form an NFAT complex with Fos and Jun. Immunoblot analyses and metabolic labeling with [32P]orthophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without affecting subcellular distribution. Dephosphorylation by in vitro treatment with calcineurin or alkaline phosphatase restores NFATp DNA binding activity and its ability to reconstitute an NFAT complex with Fos and Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA binding.

Mechanism of Action of α-Naphthoflavone as an Ah Receptor Antagonist in MCF-7 Human Breast Cancer Cells

α-Naphthotlavone (αNF) and 6-methyl-1,3,8-trichlorodiben-zofuran (MCDF) inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 gene expression in MCF-7 human breast cancer cells and also decreased the accumulation of the nuclear [3H]TCDD-aryl hydrocarbon (Ah) receptor complex. Nuclear extracts from cells treated with 10−6 M αNF and incubated with a dioxin responsive element (DRE, 26-mer) did not form a retarded band in a gel mobility shift assay. In contrast, incubation of nuclear extracts from cells treated with 10−6 M MCDF and DRE gave a retarded band and this is consistent with the antiestrogenic and Ah receptor agonist activity of MCDF in human breast cancer cells. αNF was further investigated as an Ah receptor antagonist by determining the inhibition by αNF of TCDD-induced antiestrogenicity in MCF-7 cells. TCDD (10−9 M) inhibited the 17β-estradiol-induced proliferation of MCF-7 cells and the secretion of the 52-kDa protein. In cotreatment studies, αNF (10−8 to 10−6 M) caused a concentration-dependent decrease in the antiestrogenic responses elicited by TCDD. In addition, αNF inhibited the TCDD-induced down-regulation of nuclear estrogen receptor levels in MCF-7 cells. αNF (10−6 M) alone was inactive as an estrogen or antiestrogen and in cotreatment studies did not affect 17β-estradiol-induced responses in MCF-7 cells. Tamoxifen (10−7 M), an antiestrogen which acts through the estrogen receptor, also inhibited 17β-estradiol-induced cell proliferation and aNF did not affect the tamoxifen-mediated antiproliferative response. Thus, aNF antagonized TCDD-induced CYP1A1 gene expression in MCF-7 cells and also acted as an anti-antiestrogen for TCDDmediated antiestrogemcity in these cells. These results were consistent with the low levels of DRE binding observed with nuclear extracts from cells treated with 10−9 M TCDD plus αNF (10−8 to 10−6 M) or 10−6 M αNF alone. Thus, αNF appears to act as an Ah receptor antagonist in MCF-7 cells by decreasing the levels of transcriptionally active nuclear Ah receptor complexes.

Infection of HeLa cells with poliovirus results in modification of a complex that binds to the rRNA promoter.

In HeLa cells, RNA polymerase I (Pol I)-mediated transcription is severely inhibited soon after infection with poliovirus. We have developed a gel retardation assay to analyze DNA-protein complexes formed at the Pol I promoter. We show here that two complexes (A and C) formed by nuclear extracts from uninfected cells disappear after infection of cells with poliovirus. In contrast, a new, rapidly migrating complex (D) is formed in virus-infected cell extract. This change in the mobility of gel-retarded complexes correlates well with the kinetics of inhibition of rRNA transcription in virus-infected cells. Incubation of nuclear extracts from mock-infected cells with bacterially expressed, purified poliovirus protease 3C results in the disappearance of complexes A and C with concomitant generation of complex D. A partially purified transcription factor fraction derived from uninfected cells that contains complex A is able to restore Pol I transcription when added to virus-infected cell extracts, suggesting that this complex plays an important role in Pol I transcription. These results suggest that poliovirus proteinase 3C may have an important role in the shutoff of Pol I transcription in cells infected with poliovirus.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) is an antiestrogen in human and rodent cancer cell lines: Evidence for the role of the Ah receptor

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) is a relatively nontoxic analog of 2,3,7,8-tetrachlorodibenzo- p-dioxin. Treatment of aryl hydrocarbon (Ah)-responsive MCF-7 human breast cancer cells with 100 n m MCDF resulted in the inhibition of 17β-estradiol-induced proliferation and the secretion of the 34-, 52-, and 160-kDa proteins. After treatment of the cells with 17β-[ 3H]estradiol, 100 n m of MCDF caused a decrease in the accumulation of the radiolabeled nuclear estrogen receptor (ER) complex in these cells. In parallel experiments, the antiestrogenic effects of MCDF were also determined in Ah-responsive wild-type Hepa 1c1c7 cells and Ah-nonresponsive class 1 and class 2 mutant cells. Treatment of the wild-type cells with 17β-[ 3H]estradiol and 100 n m MCDF caused a decrease in the accumulation of radiolabeled nuclear ER complex in these cells whereas no significant effects were observed in the mutant cells as determined by velocity sedimentation analysis. Comparable results were obtained using ER antibodies to measure the decrease in immunoreactive nuclear ER. In addition, both actinomycin D and cycloheximide inhibited the MCDF-mediated decrease of nuclear ER levels in the Hepa 1c1c7 wild-type cells. Although 100 n m MCDF did not induce cytochrome P-450-dependent monooxygenases in the MCF-7 or Hepa 1c1c7 cell lines, incubation of nuclear extracts from the MCF-7 cells treated with 100 n m MCDF with a synthetic consensus dioxin responsive element (an oligonucleotide duplex of 26 bases) gave a retarded band in a gel-retardation assay. The data suggest that the antiestrogenic effects of MCDF does not require the induction of the CYP1A1 gene expression but may involve the induction of other genes.

LR1, a lipopolysaccharide-responsive factor with binding sites in the immunoglobulin switch regions and heavy-chain enhancer.

In nuclear extracts of primary murine B lymphocytes cultured with LPS we have identified an inducible DNA-binding activity that is a candidate regulator of isotype-switch recombination. This LPS-responsive factor, which we refer to as LR1, is induced in LPS-cultured primary cells with kinetics that parallel isotype-switch recombination. LR1 binds sequences from the S gamma 1, S gamma 3, and S alpha switch regions, as well as the heavy-chain enhancer, and these binding sites define a consensus that occurs in each of the murine switch regions. LR1 activity is present in pre-B and B-cell lines but absent from primary B cells that have not been cultured with mitogen and from highly differentiated B-cell lines. LR1-binding activity depends on phosphorylation and is lost following incubation of nuclear extracts with acid phosphatase. The LPS inducibility and phosphorylation dependence of LR1 activity suggest that this factor monitors kinase-dependent events in cell development and communicates them to the chromosome. The locations of its binding sites and the kinetics of its induction are consistent with a role for LR1 in regulation of isotype switching.

Reversible inhibition of a thyroid-specific trans-acting factor by Ras.

Exposure of rat thyroid cells for 1 week to a temperature-sensitive variant of Kirsten murine sarcoma virus (KiMSV) Ras inactivated the thyroglobulin promoter (pTg). Cellular dedifferentiation was paralleled by the loss of the thyroid-specific trans-acting factor, TgTF1, which binds to pTg. When Ras was denatured by shifting cells to 39 degrees C, TgTF1 binding and pTg function recovered rapidly without the synthesis of new protein. TgTF1 could be reactivated in vitro by treating nuclear extracts with protein kinase A. After 4 weeks of exposure to the oncogene, denaturation of Ras no longer restored TgTF1 binding or reactivated pTg. Incubation of nuclear extracts with protein kinase A likewise did not reactivate TgTF1. Cells chronically exposed to Ras did, however, yield differentiated clones after treatment with 5-azacytidine. We suggest that Ras induces dedifferentiation in two sequential steps: (1) Ras reduces PKA activity; TgTF1 (or an auxiliary protein) becomes dephosphorylated, and binding to pTg is abolished. (2) The effects of Ras become imprinted by methylation, possibly of the TgTF1 gene.

Thyroid Hormone Receptors Form Distinct Nuclear Protein-Dependent and Independent Complexes with a Thyroid Hormone Response Element

We have examined the binding of nuclear proteins and recombinant thyroid hormone receptors (TRs) to the palindromic thyroid hormone responsive element AGGTCATGACCT (TREp) using a gel electrophoretic mobility shift assay. Four specific protein-DNA complexes were detected after incubation of nuclear extracts (NE) from T3-responsive pituitary (GH3) cells with a TREp-containing DNA fragment. This was compared with the TREp binding of reticulocyte lysate-synthesized TRs. TR alpha 1 and TR beta 2 each formed a single major TR:TREp complex which comigrated with the least retarded complex formed by GH3 NE, while TR beta 1 formed multiple complexes suggesting that it can bind to TREp as an oligomer. Interestingly, coincubation of 35S-TR alpha 1, GH3 NE, and unlabeled TREp resulted in not only the 35S-TR:TREp complex, but in two additional more greatly retarded complexes containing 35S-TR alpha 1 and comigrating with those formed by GH3 extract alone. Incubation of each of the TRs with NE from COS-7 cells, which do not possess sufficient endogenous TRs to mediate T3-responses, resulted in formation of a new, more greatly shifted complex. A similar, heat labile activity which altered mobility of the TR:TRE complex was also present in NE from T3-unresponsive JEG-3 cells. At high concentration of NE, all of the TR bound to TREp was more greatly retarded than in the absence of NE. Truncation of TR alpha 1 at amino acid 210 prevented additional complex formation in the presence of NE without affecting DNA binding, suggesting that the carboxyl-terminus of the TRs is essential for interaction with nuclear proteins.(ABSTRACT TRUNCATED AT 250 WORDS)