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Transient Transfection (transient + transfection)
Terms modified by Transient Transfection Selected AbstractsDown-regulation of heme oxygenase-2 is associated with the increased expression of heme oxygenase-1 in human cell linesFEBS JOURNAL, Issue 23 2006Yuanying Ding Intracellular heme concentrations are maintained in part by heme degradation, which is catalyzed by heme oxygenase. Heme oxygenase consists of two structurally related isozymes, HO-1 and HO-2. Recent studies have identified HO-2 as a potential oxygen sensor. To gain further insights into the regulatory role of HO-2 in heme homeostasis, we analyzed the expression profiles of HO-2 and the biochemical consequences of HO-2 knockdown with specific short interfering RNA (siRNA) in human cells. Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and HepG2 hepatoma. Down-regulation of HO-2 expression with siRNA against HO-2 (siHO-2) caused induction of HO-1 expression at both mRNA and protein levels in HeLa and HepG2 cells. In contrast, knockdown of HO-1 expression did not noticeably influence HO-2 expression. HO-2 knockdown prolonged the half-life of HO-1 mRNA twofold in HeLa cells. Transient transfection assays in HeLa cells revealed that the 4.5-kb human HO-1 gene promoter was activated with selective knockdown of HO-2 in a sequence-dependent manner. Moreover, HO-2 knockdown caused heme accumulation in HeLa and HepG2 cells only when exposed to exogenous hemin. HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellular heme homeostasis. These results suggest that HO-2 may down-regulate the expression of HO-1, thereby directing the co-ordinated expression of HO-1 and HO-2. [source] Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activityFEBS JOURNAL, Issue 15 2002Hannes E. Bülow In this study we describe the isolation of three genes of the CYP11B family of the guinea pig. CYP11B1 codes for the previously described 11,-hydroxylase [Bülow, H.E.,Möbius, K., Bähr, V. & Bernhardt, R. (1996) Biochem. Biophys. Res. Commun. 221, 304,312] while CYP11B2 represents the aldosterone synthase gene. As no expression for CYP11B3 was detected this gene might represent a pseudogene. Transient transfection assays show higher substrate specificity for its proper substrate for CYP11B1 as compared to CYP11B2, which could account for the zone-specific synthesis of mineralocorticoids and glucocorticoids, respectively. Thus, CYP11B2 displayed a fourfold higher ability to perform 11,-hydroxylation of androstenedione than CYP11B1, while this difference is diminished with the size of the C17 substituent of the substrate. Furthermore, analyses with the electron transfer protein adrenodoxin indicate differential sensitivity of CYP11B1 and CYP11B2 as well as the three hydroxylation steps catalysed by CYP11B2 to the availability of reducing equivalents. Together, both mechanisms point to novel protein intrinsic modalities to achieve tissue-specific production of mineralocorticoids and glucocorticoids in the guinea pig. In addition, we conducted phylogenetic analyses. These experiments suggest that a common CYP11B ancestor gene that possessed both 11,-hydroxylase and aldosterone synthase activity underwent a gene duplication event before or shortly after the mammalian radiation with subsequent independent evolution of the system in different lines. Thus, a differential mineralocorticoid and glucocorticoid synthesis might be an exclusive achievement of mammals. [source] MITF-CM, a newly identified isoform of microphthalmia-associated transcription factor, is expressed in cultured mast cellsINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 2 2009M. SHIOHARA Summary The microphthalmia-associated transcription factor (MITF) gene encodes a basic helix-loop-helix and leucin zipper protein. In this study, we identified a novel MITF isoform, MITF-CM, which possesses a unique amino terminus. Exon 1CM is located 84 kb upstream of the exon encoding the B1b domain. MITF-CM was expressed in the human mast cell line HMC-1, the human basophilic cell line KU812, and CB-derived mast cells cultured for 10 weeks as well as bone marrow mononuclear cells. Transient transfection of MITF-CM cDNA in COS-7 cells resulted in the expression of a 64-kDa protein, detected by Western blotting, and nuclear localization of the protein, detected by immunostaining. The transient cotransfection of a luciferase construct under the control of the tyrosinase promoter and MITF-CM cDNA increased luciferase activity threefold. In contrast, none of the MITF isoforms transactivated both the tryptase and chymase gene promoters, indicating differences in the gene transactivation system between humans and mice. [source] The interaction of KCTD1 with transcription factor AP-2, inhibits its transactivation,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Xiaofeng Ding Abstract AP-2 is a transcription factor implicated in mammalian development, cell proliferation, apoptosis, and carcinogenesis. To identify potential AP-2,-interacting partners, a yeast two-hybrid screen was performed in human brain cDNA library. One of the identified clones encodes potassium channel tetramerization domain-containing 1 (KCTD1). We demonstrated the novel KCTD1,AP-2, interaction in vitro by GST pull-down assays and in vivo by co-immunoprecipitation assays and mapped the interaction domains to the N-termini of both proteins. In addition, we observed that the two proteins were completely co-localized in the nuclei of mammalian cells. Transient transfection assays using four promoters containing AP-2-binding sites confirmed that KCTD1 significantly repressed AP-2,-mediated transactivation through the BTB domain, whereas KCTD1 siRNA strongly relieved KCTD1-mediated repression of AP-2, transcriptional activity, and other BTB domain proteins such as PDIP1, KCTD10, and TNFAIP1 did not markedly inhibit the transcriptional activity of AP-2,, suggesting that KCTD1 specifically acts as a negative regulator of AP-2,. Finally, we found that KCTD1 interacted with three major members of the AP-2 family and inhibited their transcriptional activities. Taken together, our results indicate the novel function of KCTD1 as the transcriptional repressor for AP-2 family, especially for AP-2,. J. Cell. Biochem. 106: 285,295, 2009. © 2008 Wiley-Liss, Inc. [source] The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Peter V.N. Bodine Abstract Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display-polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled-related protein (sFRP)-1 was the most highly regulated of these. Transient transfection of HOBs with sFRP-1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP-1 mRNA levels increased 24-fold during HOB differentiation from pre-osteoblasts to pre-osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the pre-osteocytes, which had the highest levels of sFRP-1 mRNA, also had the highest rate of cell death. Basal sFRP-1 mRNA levels also increased 29-fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP-1 mRNA was induced 38-fold following prostaglandin E2 (PGE2) treatment of pre-osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP-1 expression was down-regulated by as much as 80% following transforming growth factor (TGF)-,1 treatment of pre-osteocytes that had high basal mRNA levels. Consistent with this, treatment of pre-osteoblasts and mature osteoblasts with PGE2 increased apoptosis threefold, while treatment of pre-osteocytes with TGF-,1 decreased cell death by 50%. Likewise, over-expression of sFRP-1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP-1 as an important negative regulator of human osteoblast and osteocyte survival. © 2005 Wiley-Liss, Inc. [source] Chromium (VI) inhibits heme oxygenase-1 expression in vivo and in arsenic-exposed human airway epithelial cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2006Kimberley A. O'Hara Inhaled hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms. One hypothesis for this lung pathogenesis is that Cr(VI) silences induction of cytoprotective genes, such as heme oxygenase-1 (HO-1), whose total lung mRNA levels were reduced 21 days after nasal instillation of potassium dichromate in C57BL/6 mice. To investigate the mechanisms for this inhibition, Cr(VI) effects on basal and arsenic (As(III))-induced HO-1 expression were examined in cultured human bronchial epithelial (BEAS-2B) cells. An effect of Cr(VI) on the low basal HO-1 mRNA and protein levels in BEAS-2B cells was not detectible. In contrast, Cr(VI) added to the cells before As(III), but not simultaneously with As(III), attenuated As(III)-induced HO-1 expression. Transient transfection with luciferase reporter gene constructs controlled by the full length ho-1 promoter or deletion mutants demonstrated that this inhibition occurred in the E1 enhancer region containing critical antioxidant response elements (ARE). Cr(VI) pretreatment inhibited As(III)-induced activity of a transiently expressed reporter construct regulated by three ARE tandem repeats. The mechanism for this Cr(VI)-attenuated transactivation appeared to be Cr(VI) reduction of the nuclear levels of the transcription factor Nrf2 and As(III)-stimulated Nrf2 transcriptional complex binding to the ARE cis element. Finally, exposing cells to Cr(VI) prior to co-exposure with As(III) synergized for apoptosis and loss of membrane integrity. These data suggest that Cr(VI) silences induction of ARE-driven genes required for protection from secondary insults. The data also have important implications for understanding the toxic mechanisms of low level, mixed metal exposures in the lung. J. Cell. Physiol. 209: 113,121, 2006. © 2006 Wiley-Liss, Inc. [source] Participation of protein kinase C , isoform and extracellular signal-regulated kinase in neurite outgrowth of GT1 hypothalamic neuronsJOURNAL OF NEUROCHEMISTRY, Issue 6 2002Youngshik Choe Abstract In the present study, we investigated the selective role of protein kinase C (PKC) isoforms on neurite outgrowth of the GT1 hypothalamic neurons using several PKC isoform-selective inhibitors and transfection-based expression of enhanced green fluorescence protein (EGFP)-fused PKC isoforms. 12- O -Tetradecanoylphorbol-13-acetate (TPA) induced neurite outgrowth and growth cone formation, effects that were blocked by GF 109203X (a PKC inhibitor), safingolTM(a PKC,-selective inhibitor), but not by rottlerinTM (a PKC,-selective inhibitor), indicating that PKC, may be selectively involved in neurite outgrowth and cytoskeletal changes of filamentous actin and ,-tubulin. To define the differential localization of PKC isoforms, EGFP-tagged PKC,, PKC,, and PKC, were transfected into GT1 neuronal cells. TPA treatment induced relocalization of PKC,-EGFP to growth cones and cell,cell adhesion sites, PKC,-EGFP to the nucleus, and PKC,-EGFP to the membrane ruffle, respectively. An EGFP chimera of the catalytic domain of PKC, (PKC,-Cat-EGFP), the expression of which was inducible by doxycycline, was employed to directly ascertain the effect of PKC, enzymatic activity on neurite outgrowth of GT1 cells. Transient transfection of PKC,-Cat-EGFP alone increased the neurite-outgrowth and doxycycline treatment further augmented the number of neurite-containing cells. We also examined the involvement of the extracellular signal-regulated kinase (ERK) MAP kinase in TPA-induced neurite outgrowth. TPA treatment increased phosphorylated ERK MAP kinase, but not p38 MAP kinase. Specific inhibition of PKC, with safingol blocked the phosphorylation of ERK induced by TPA. More importantly, both neurite outgrowth and phosphorylation of ERK by TPA were blocked by PD 098059, a specific inhibitor of MEK (MAP kinase/ERK kinase-1), but not by SB203580, a specific inhibitor of p38 MAP kinase. These results demonstrate that PKC, isoform-specific activation is involved in neurite outgrowth of GT1 hypothalamic neuronal cells via ERK, but not the p38 MAP kinase signal pathway. [source] IL-1,, an immediate early protein secreted by activated microglia, induces iNOS/NO in C6 astrocytoma cells through p38 MAPK and NF-,B pathwaysJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2006Yun-Jung Kim Abstract In the present study we sought to examine cell,cell interactions by investigating the effects of factors released by stimulated microglia on inducible nitric oxide (NO) synthase (iNOS) induction in astrocytoma cells. After examining the temporal profiles of proinflammatory molecules induced by lipopolysaccharide (LPS) stimulation in BV2 microglial cells, iNOS and IL-1, were observed to be the first immediate-response molecules. Removal of LPS after 3 hr stimulation abrogated NO release, whereas a full induction of IL-1, was retained in BV2 cells. We observed consistently that conditioned medium (CM) from activated microglia resulted in the induction of iNOS in C6 cells, and IL-1, was shown to be a key regulator of iNOS induction. An IL-1,-neutralizing antibody diminished NO induction. Incubation with recombinant IL-1, stimulated NO release to a lesser extent compared to microglial CM; co-treatment of LPS and IL-1, had a potent, synergistic effect on NO release from C6 cells. Transient transfection with MEK kinase 1 (MEKK1) or nuclear factor-kappa B (NF-,B) expression plasmids induced iNOS, and IL-1, further enhanced the MEKK1 response. Furthermore, IL-1,-mediated NO release from C6 cells was significantly suppressed by inhibition of p38 mitogen activated protein kinase (MAPK) or NF-,B by specific chemical inhibitors. Both IL-1, and MEKK1 stimulated p38 and JNK MAPKs, as well as the NF-,B pathway, to induce iNOS in C6 cells. Microglia may represent an anti-tumor response in the central nervous system, which is potentiated by the local secretion of immunomodulatory factors that in turn affects astrocytoma (glioma) cells. A better understanding of microglia,glioma or microglia,astrocyte interactions will help in the design of novel immune-based therapies for brain tumors or neuronal diseases. © 2006 Wiley-Liss, Inc. [source] Ethanol-Induced Up-Regulation of the Urokinase Receptor In Cultured Human Endothelial CellsALCOHOLISM, Issue 2 2001Edlue M. Tabengwa Background: Moderate alcohol consumption has been correlated to reduced coronary artery disease (CAD) risk and mortality. This alcohol effect may be mediated in part by an increased endothelial cell (EC) fibrinolysis. ECs synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase type plasminogen activator (u-PA), and plasminogen activator inhibitor type-1(PAI-1). In addition, they synthesize and regulate receptors for fibrinolytic proteins, namely (t-PA and plasminogen receptor) Annexin II and u-PA receptor (u-PAR). These receptors play an important role in the regulated expression of receptor-bound plasminogen activator conversion of receptor-bound plasminogen to receptor-bound plasmin on the EC surface (surface-localized fibrinolytic activity). Therefore, systemic factors, such as ethanol, that affect the level, or activity or interaction of one or more of these components, resulting in the increased expression of surface-localized EC fibrinolytic activity, will be expected to reduce the risk for thrombosis, CAD, and myocardial infarction (MI). We have previously shown that low ethanol up-regulates t-PA and u-PA gene transcription, while it down-regulates PAI-1, hence resulting in increased (sustained, 24 hr) surface-localized EC fibrinolytic activity. The current studies were carried out to determine whether low ethanol increased u-PAR expression in cultured human umbilical cord vein ECs (HUVECs). Methods: Cultured HUVECs were preincubated (1 hr) in the absence/presence of ethanol (0.025,0.2%, v/v); u-PAR mRNA (RT-PCR), antigen (western blot), and activity (125I-u-PA ligand binding/Scatchard analysis) levels were then measured after 0,24 hr. To determine whether the ethanol-induced changes in the u-PAR expression were transcriptional, transient transfection studies were carried out using a u-PAR/luciferase promoter construct (pu-PAR120/luc [1.2-kb u-PAR promoter fragment ligated to a promoterless luciferase vector]). Results: uPAR mRNA levels increased 2- to 3-fold and antigen levels (western blot) increased 2- to 4-fold while u-PA binding activity increased 36% (1.25 vs. 1.7 , 105 sites/cell, Bmax) without significantly affecting the Kd (1,2 nM). Transient transfection of cultured HUVECs with a pu-PAR120/luc construct resulted in a 2- to 3-fold increase in promoter activity in ethanol-induced cultures, compared with controls. Conclusion: These combined results demonstrate that low ethanol (,0.1%, v/v) induces the up-regulation of u-PAR gene transcription, resulting in increased u-PAR ligand binding activity. These results also further identify/define the contribution and role of another fibrinolytic protein in the overall ethanol-induced increase in surface-localized EC fibrinolysis that may underlie and contribute, in part, to the cardioprotection attributed to moderate alcohol consumption. [source] The voltage-dependent Cl, channel ClC-5 and plasma membrane Cl, conductances of mouse renal collecting duct cells (mIMCD-3)THE JOURNAL OF PHYSIOLOGY, Issue 3 2001J. A. Sayer 1We have tested the hypothesis that the voltage-dependent Cl, channel, ClC-5 functions as a plasma membrane Cl, conductance in renal inner medullary collecting duct cells. 2Full-length mouse kidney ClC-5 (mClC-5) was cloned and transiently expressed in CHO-K1 cells. Fast whole-cell patch-clamp recordings confirmed that mClC-5 expression produces a voltage-dependent, strongly outwardly rectifying Cl, conductance that was unaffected by external DIDS. 3Slow whole-cell recordings, using nystatin-perforated patches from transfected CHO-K1 cells, also produced voltage-dependent Cl, currents consistent with ClC-5 expression. However, under this recording configuration an endogenous DIDS-sensitive Ca2+ -activated Cl, conductance was also evident, which appeared to be activated by green fluorescent protein (GFP) transfection. 4A mClC-5-GFP fusion protein was transiently expressed in CHO-K1 cells; confocal laser scanning microscopy (CLSM) showed localization at the plasma membrane, consistent with patch-clamp experiments. 5Endogenous expression of mClC-5 was demonstrated in mouse renal collecting duct cells (mIMCD-3) by RT-PCR and by immunocytochemistry. 6Using slow whole-cell current recordings, mIMCD-3 cells displayed three biophysically distinct Cl, -selective currents, which were all inhibited by DIDS. However, no cells exhibited whole-cell currents that had mClC-5 characteristics. 7Transient transfection of mIMCD-3 cells with antisense mClC-5 had no effect on the endogenous Cl, conductances. Transient transfection with sense mClC-5 failed to induce the Cl, conductance seen in CHO-K1 cells but stimulated levels of the endogenous Ca2+ -activated Cl, conductance 24 h post-transfection. 8Confocal laser scanning microscopy of mIMCD-3 cells transfected with mClC-5-GFP showed that the protein was absent from the plasma membrane and was instead localized to acidic endosomal compartments. 9These data discount a major role for ClC-5 as a plasma membrane Cl, conductance in mIMCD-3 cells but suggest a role in endosomal function. [source] Modulation of the angiogenic phenotype of normal and systemic sclerosis endothelial cells by gain,loss of function of pentraxin 3 and matrix metalloproteinase 12ARTHRITIS & RHEUMATISM, Issue 8 2010Francesca Margheri Objective Studies have shown that in systemic sclerosis (SSc) endothelial cells, overproduction of matrix metalloproteinase 12 (MMP-12) and pentraxin 3 (PTX3) is associated with defective angiogenesis. This study was undertaken to examine whether overexpression of the relevant molecules could inhibit angiogenesis of normal microvascular endothelial cells (MVECs), and whether silencing of these molecules in SSc MVECs could restore the lost angiogenic properties of the cells in vitro and in vivo. Methods Transient transfection of MVECs with human MMP12 and PTX3 was performed by electroporation. Silencing of MMP12 and PTX3 was obtained by treatment with small interfering RNA, and treatment effects were validated by Western blotting with specific antibodies and a fluorimetric assay. In vitro cell migration and capillary morphogenesis were studied on Matrigel substrates. In vivo angiogenesis was studied using a Matrigel sponge assay in mice. Results Transfection of MMP12 and PTX3 in normal MVECs resulted in loss of proliferation, invasion, and capillary morphogenesis in vitro, attributed to truncation of the urokinase-type plasminogen activator receptor by MMP12 and to the anti,fibroblast growth factor 2/anti,vascular endothelial growth factor activity of PTX3. These effects were particularly evident in mixed populations of transfected normal MVECs (50% transfected with MMP12 and 50% with PTX3). Silencing of the same molecules in SSc MVECs increased their invasion in Matrigel. Single-gene silencing did not increase the capillary morphogenesis of SSc MVECs, whereas double-gene,silenced cells showed a burst of capillary tube formation. Culture medium of silenced SSc MVECs stimulated angiogenesis in assays of Matrigel sponge invasion in mice. Conclusion Overexpression of either MMP12 or PTX3 in normal MVECs blunts their angiogenic properties. Loss of function of MMP12 and PTX3 in SSc MVECs restores the ability of the cells to produce capillaries in vitro and induces vascularization in vivo on a Matrigel sponge. [source] Mediation of interleukin-1,,induced transforming growth factor ,1 expression by activator protein 4 transcription factor in primary cultures of bovine articular chondrocytes: Possible cooperation with activator protein 1ARTHRITIS & RHEUMATISM, Issue 6 2003R. Andriamanalijaona Objective Interleukin-1 (IL-1) and transforming growth factor ,1 (TGF,1) play major roles in osteoarticular diseases, exerting opposite effects on both the catabolism and anabolism of cartilage matrix. Previous findings suggest that IL-1 and TGF,1 could function in a feedback interaction. However, the effect exerted by IL-1 on expression of TGF, by articular chondrocytes is, so far, poorly understood. The present study was carried out to determine the influence of IL-1, on the expression of TGF,1 by bovine articular chondrocytes (BACs) in primary culture. Methods BAC primary cultures were treated with IL-1,, and TGF,1 messenger RNA (mRNA) steady-state levels and protein expression were measured by real-time reverse transcription,polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Transient transfection of TGF,1 gene promoter constructs was performed to delineate the DNA sequences that mediate the IL-1, effect. Electrophoretic mobility shift assays (EMSAs) and supershift analysis were used to characterize the transcription factors binding to these sequences. Results Cultured BACs responded to IL-1, exposure by exhibiting an increase of TGF,1 expression at both the mRNA and protein levels. The effect was found to be mediated by a major 80-bp sequence located between ,732 and ,652 upstream of the transcription initiation site. EMSA and supershift analysis revealed that the transcription factors activator protein 4 (AP-4) and AP-1 specifically bound to the ,720/,696 part of this sequence under IL-1, treatment. Overexpression of AP-4 in the BAC cultures resulted in stimulation of the transcriptional activity of the ,732/+11 TGF,1 promoter construct through the same IL-1,,responsive element. Conclusion IL-1, induces an increase of TGF,1 in articular chondrocytes through activation of AP-4 and AP-1 binding to the TGF,1 gene promoter. These findings may help us understand the role of IL-1, in the disease process. Notwithstanding its deleterious effect on cartilage, IL-1 could initiate the repair response displayed by injured cartilage in the early stages of osteoarthritis through its ability to enhance TGF,1 expression by local chondrocytes. [source] Sodium butyrate induces P53-independent, Fas-mediated apoptosis in MCF-7 human breast cancer cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2002Valérie Chopin This study was performed to determine the effect and action mechanisms of sodium butyrate (NaB) on the growth of breast cancer cells. Butyrate inhibited the growth of all breast cancer cell lines analysed. It induced cell cycle arrest in G1 and apoptosis in MCF-7, MCF-7ras, T47-D, and BT-20 cells, as well as arrest in G2/M in MDA-MB-231 cells. Transient transfection of MCF-7 and T47-D cells with wild-type and antisense p53 did not modify butyrate-induced apoptosis. Pifithrin-,, which inhibits the transcriptional activity of P53, did not modify cell growth or apoptosis of MCF-7 and T47-D cells treated with butyrate. These results indicate that P53 was not involved in butyrate-induced growth inhibition of breast cancer cells. Treatment of MCF-7 cells with anti-Fas agonist antibody induced cell death, indicating that Fas was functional in these cells. Moreover, butyrate potentiated Fas-induced apoptosis, as massive apoptosis was observed rapidly when MCF-7 cells were treated with butyrate and anti-Fas agonist antibody. In addition, butyrate-induced apoptosis in MCF-7 cells was considerably reduced by anti-Fas antagonist antibody. Western blot analysis showed that butyrate increased Fas and Fas ligand levels (Fas L), indicating that butyrate-induced apoptosis may be mediated by Fas signalling. These results demonstrate that butyrate inhibited the growth of breast cancer cells in a P53-independent manner. Moreover, it induced apoptosis via the Fas/Fas L system and potentiated Fas-triggered apoptosis in MCF-7 cells. These findings may open interesting perspectives in human breast cancer treatment strategy. British Journal of Pharmacology (2002) 135, 79,86; doi:10.1038/sj.bjp.0704456 [source] Parathyroid hormone inhibits phosphorylation of mitogen-activated protein kinase (MAPK) ERK1/2 through inhibition of c-Raf and activation of MKP-1 in osteoblastic cellsCELL BIOCHEMISTRY AND FUNCTION, Issue 5 2009Lick Pui Lai Abstract Parathyroid hormone (PTH) regulation of mitogen-activated protein kinases (MAPK) ERK1/2 contributes to PTH regulation of osteoblast growth and apoptosis. We investigated the mechanisms by which PTH inhibits ERK1/2 activity in osteoblastic UMR 106-01 cells. Treatment with PTH significantly inhibited phosphorylated ERK1/2 between 5 and 60,min. Transient transfection of cells with a cDNA encoding MAPK phosphatase-1 (MKP-1) resulted in 30,40% inhibition of pERK1/2; however MKP-1 protein levels were only significantly stimulated by PTH after 30,mins, suggesting another mechanism for the early phase of pERK1/2 inhibition. The active upstream kinase c-Raf phosphorylation at serine 338 (ser338) was significantly inhibited by PTH treatment within 5,min and transfection of the cells with constitutively-active c-Raf blocked PTH inhibition of pERK1/2. Inhibition of pERK1/2 and phosphor-c-Raf were seen when cells were treated with PTH(1-34) or PTH(1-31) analogues that stimulate cAMP, but not with PTH(3-34), PTH(7-34) or PTH(18-48) that do not stimulate cAMP. Stimulation of the cells with forskolin or 8BrcAMP also inhibited pERK1/2 and c-Raf.p338. Our results suggest that rapid PTH inhibition of ERK1/2 activity is mediated by PKA dependent inhibition of c-Raf activity and that stimulation of MKP-1 may contribute to maintaining pERK1/2 inhibition over prolonged time. Copyright © 2009 John Wiley & Sons, Ltd. [source] Transient transfection of epidermal growth factor receptor gene into MCF7 breast ductal carcinoma cell lineCELL BIOCHEMISTRY AND FUNCTION, Issue 3 2005Majed S. Alokail Abstract Epidermal growth factor receptor (EGFR) is activated by autocrine growth factors in many types of tumours, including breast tumours. This receptor has been linked to a poor prognosis in breast cancer and may promote proliferation, migration, invasion, and cell survival as well as inhibition of apoptosis. Human breast ductal carcinoma MCF7 cells were transfected using FuGENEÔ 6 with 1,,g of pcDNA3-EGFR containing the full-length human EGFR promoter or 1,,g of the vectors alone (pcDNA3). The transfected cells were transferred into a 25-cm2 flask containing growth medium and G418. Confluent cultures were lysed, total protein levels measured and electrophoresed. The electrophoresed samples were transferred to nitrocellulose and incubated overnight at 4°C with either anti-EGFR or anti-phospho-ERK and immunoreactive bands were visualized using HRP-linked secondary antibody. We created a model system of EGFR overexpression in MCF7 clones with stably transfected pcDNA3/EGFR plasmid. These cells have been shown to promote substantial phosphorylation of both ERK1 and ERK2. The high level of EGFR and ERK1/2 phosphorylation was not seen in the pcDNA3 vector control cells or in non-transfected cells. In this article we describe successful transient transfection experiments on MCF7 cells using the FuGENEÔ 6 Transfection Reagent. The overexpression of EGFR could be a mediated stress response and a survival signal that involves ERK1 and ERK2 phosphorylation. Copyright © 2004 John Wiley & Sons, Ltd. [source] Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR geneFEBS JOURNAL, Issue 2 2002Marios Phylactides The cystic fibrosis transmembrane conductance regulator (CFTR) gene shows a complex pattern of expression, with temporal and spatial regulation that is not accounted for by elements in the promoter. One approach to identifying the regulatory elements for CFTR is the mapping of DNase I hypersensitive sites (DHS) within the locus. We previously identified at least 12 clusters of DHS across the CFTR gene and here further evaluate DHS in introns 2, 3, 10, 16, 17a, 18, 20 and 21 to assess their functional importance in regulation of CFTR gene expression. Transient transfections of enhan- cer/reporter constructs containing the DHS regions showed that those in introns 20 and 21 augmented the activity of the CFTR promoter. Structural analysis of the DNA sequence at the DHS suggested that only the one intron 21 might be caused by inherent DNA structures. Cell specificity of the DHS suggested a role for the DHS in introns 2 and 18 in CFTR expression in some pancreatic duct cells. Finally, regulatory elements at the DHS in introns 10 and 18 may contribute to upregulation of CFTR gene transcription by forskolin and mitomycin C, respectively. These data support a model of regulation of expression of the CFTR gene in which multiple elements contribute to tightly co-ordinated expression in vivo. [source] Polyphenolics Increase t-PA and u-PA Gene Transcription in Cultured Human Endothelial CellsALCOHOLISM, Issue 2 2001Laila H. Abou-Agag Background: Moderate red wine consumption has been associated with a reduced risk for coronary heart disease, and this cardioprotection may be mediated, in part, by promoting fibrinolysis. This protection may be attributed to the combined or perhaps synergistic effects of alcohol and other red wine components (i.e., polyphenolics). These studies were carried out to determine whether individual phenolics (i.e., catechin, epicatechin, quercetin, and resveratrol) affect fibrinolytic protein (tissue-type plasminogen activator [t-PA] and urokinase-type PA [u-PA]) e-pression and surface-localized fibrinolytic activity in cultured human umbilical vein endothelial cells (HUVECs). Methods: Cultured HUVECs were preincubated (1 hr, 37°C) in the absence or presence of varying concentrations of catechin, epicatechin, quercetin, and resveratrol (0.001,10 ,M) and then were washed and incubated for various times in the absence of phenolics. Secreted t-PA/u-PA antigen (24 hr, enzyme-linked immunoadsorbent assay) and mRNA [0,16 hr, reverse transcription-polymerase chain reaction(RT-PCR)] levels and fibrinolytic activity (direct activation of HUVEC-bound 125I-labeled glutamyl-plasminogen, quantitation of 125I-labeled M r 20 kDa plasmin light-chain) were measured. Transient transfections of cultured HUVECs were carried out with the pt-PA222/luc and pu-PA236/luc promoter constructs, by using lipofectamine. Results: Each of the phenolics similarly increased t-PA and u-PA antigen (2- to 3-fold) and mRNA (3- to 4-fold) levels, concomitant with an increase (2- to 3-fold) in sustained (24 hr), surface-localized fibrinolytic activity. Transcription inhibitor actinomycin D abolished the induction of t-PA and u-PA mRNA e-pression by these phenolics. Transfections with the pt-PA222/luc and pu-PA236/luc promoter constructs showed 2- to 3-fold and 2- to 4-fold increases in luciferase activity for t-PA and u-PA, respectively. Conclusions: These results demonstrate that each of these phenolics up-regulates both t-PA and u-PA gene transcription, which results in the sustained increased e-pression of surface-localized fibrinolytic activity in cultured HUVECs. Wine phenolics increase fibrinolytic activity, independent of ethanol, and it is likely that the overall cardioprotective benefits associated with moderate red wine consumption are attributable to the combined, additive, or perhaps synergistic effects of alcohol and other wine components. [source] A designed curved DNA segment that is a remarkable activator of eukaryotic transcriptionFEBS JOURNAL, Issue 24 2006Noriyuki Sumida To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase (tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system, and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. Interestingly, T20 was found to activate transcription in all the stable transformants, irrespective of the locus. This suggests that the T20 segment may allow stable expression of transgenes, which is of importance in many fields, and may also be useful for the construction of nonviral vectors for gene therapy. [source] Differential effects of histone deacetylase inhibitors on phorbol ester- and TGF-,1 induced murine tissue inhibitor of metalloproteinases-1 gene expressionFEBS JOURNAL, Issue 8 2005David A. Young Expression of the tissue inhibitor of metalloproteinases-1 (Timp-1) gene can be induced by either phorbol myristate acetate (PMA) or transforming growth factor ,1 (TGF-,1), although the signalling pathways involved are not clearly defined. Canonically, histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) or sodium butyrate (NaB) increase total cellular histone acetylation and activate expression of susceptible genes. Remarkably, PMA and TGF-,1 stimulation of Timp-1 show a differential response to TSA or NaB. TSA or NaB potentiate PMA-induced Timp-1 expression but repress TGF-,1-induced Timp-1 expression. The repression of TGF-,1-induced Timp-1 by TSA was maximal at 5 ng·mL,1, while for the superinduction of PMA-induced Timp-1 expression, the maximal dose is >,500 ng·mL,1 TSA. A further HDACi, valproic acid, did not block TGF-,1-induced Timp-1 expression, demonstrating that different HDACs impact on the induction of Timp-1. For either PMA or TGF-,1 to induce Timp-1 expression, new protein synthesis is required, and the induction of AP-1 factors closely precedes that of Timp-1. The effects of the HDACi can be reiterated in transient transfection using Timp-1 promoter constructs. Mutation or deletion of the AP-1 motif (,59/,53) in the Timp-1 promoter diminishes PMA-induction of reporter constructs, however, the further addition of TSA still superinduces the reporter. In c-Jun,/, cells, PMA still stimulates Timp-1 expression, but TSA superinduction is lost. Transfection of a series of Timp-1 promoter constructs identified three regions through which TSA superinduces PMA-induced Timp-1 and we have demonstrated specific protein binding to two of these regions which contain either an avian erythroblastosis virus E26 (v-ets) oncogene homologue (Ets) or Sp1 binding motif. [source] Identification of proNeuropeptide FFA peptides processed in neuronal and non-neuronal cells and in nervous tissueFEBS JOURNAL, Issue 20 2003Elisabeth Bonnard Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in a neuronal (human neuroblastoma SH-SY5Y) cell line and in COS-7 cells after transient transfection of the human proNPFFA cDNA and were compared with those detected in the mouse spinal cord. After reverse-phase high performance liquid chromatography of soluble material, NPFF-related peptides were immunodetected with antisera raised against NPFF and identified by using on-line capillary liquid chromatography/nanospray ion trap tandem mass spectrometry. Neuronal and non-neuronal cells generated different peptides from the same precursor. In addition to NPFF, SQA-NPFF (Ser-Gln-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPAF were identified in the human neuroblastoma while only NPFF was clearly identified in COS-7 cells. In mouse, in addition to previously detected NPFF and NPSF, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide), the homologous peptide of SQA-NPFF, were characterized. These data on intracellular processing of proNeuropeptide FFA are discussed in regard to the known enzymatic processing mechanisms. [source] Sp1 and Sp3 are involved in up-regulation of human deoxyribonuclease II transcription during differentiation of HL-60 cellsFEBS JOURNAL, Issue 8 2003San-Fang Chou Expression of DNase II in macrophages is potentially crucially important in the removal of unwanted DNA. We have previously shown that DNase II expression is up-regulated at the transcriptional level during the phorbol 12-myristate-13-acetate (PMA)-induced differentiation of HL-60 and THP-1 cells. In this study, we investigated the cis -regulatory elements and transcription factors involved in this process in HL-60 cells. cis -Regulatory elements in the DNase II promoter were located by 5, deletion and site-directed mutagenesis of promoter-luciferase constructs and transient transfection of HL-60 cells. Furthermore, the binding proteins were identified by electrophoretic mobility shift assay (EMSA) in the presence of specific antibodies. In the DNase II promoter, 249 base pairs upstream of the transcription start site were essential for maximal promoter activity in both untreated and PMA-treated HL-60 cells and, within this region, three Sp1 and Sp3 binding sites were identified as essential for transcriptional regulation and PMA induction. Western blot analysis showed that PMA treatment resulted in increased levels of Sp1 and Sp3 proteins. Furthermore, cotransfection analysis in Drosophila SL2 cells showed that Sp1 was more potent than Sp3 in activating the DNase II promoter. We therefore conclude that Sp1 and/or Sp3 are involved in the up-regulation of DNase II expression during the differentiation of HL-60 cells. [source] Hormonal regulation of multiple promoters of the rat mitochondrial glycerol-3-phosphate dehydrogenase geneFEBS JOURNAL, Issue 14 2001Identification of a complex hormone-response element in the ubiquitous promoter B Rat mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is regulated by multiple promoters in a tissue-specific manner. Here, we demonstrate that thyroid hormone (3,5,3,-tri-iodo- l -thyronine) and steroid hormone but not the peroxisome proliferator clofibrate and retinoic acid stimulate the activation of the ubiquitous promoter B in a receptor-dependent manner, whereas the more tissue-restricted promoters A and C are not inducible by these hormones. Thyroid hormone action is mediated by a direct repeat +4 (DR+4) hormone-response element as identified by deletion and mutation analyses of promoter B in transient transfection analyses. The DR+4 element was able to bind to an in vitro translated thyroid hormone receptor in band-shift and supershift experiments. The hormone-response element comaps with a recognition site for the transcription factor Sp1, suggesting complex regulation of this sequence element. Mutation of this Sp1-recognition site reduces the basal promoter B activity dramatically in HepG2 and HEK293 cells in transient transfection and abolishes the binding of Sp1 in band-shift experiments. As demonstrated by Western-blot experiments, administration of tri-iodothyronine to euthyroid rats increases hepatic mGPDH protein concentrations in vivo. As it has recently been reported that human mGPDH promoter B is not regulated by tri-iodothyronine, this is the first example of a differentially tri-iodothyronine-regulated orthologous gene promoter in man and rat. [source] Phytanoyl-CoA hydroxylase activity is induced by phytanic acidFEBS JOURNAL, Issue 13 2000Anna W. M. Zomer Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid present in various dietary products such as milk, cheese and fish. In patients with Refsum disease, accumulation of phytanic acid occurs due to a deficiency of phytanoyl-CoA hydroxylase, a peroxisomal enzyme containing a peroxisomal targeting signal 2. Recently, phytanoyl-CoA hydroxylase cDNA has been isolated and functional mutations have been identified. As it has been shown that phytanic acid activates the nuclear hormone receptors peroxisome proliferator-activated receptor (PPAR), and all three retinoid X receptors (RXRs), the intracellular concentration of this fatty acid should be tightly regulated. When various cell lines were grown in the presence of phytanic acid, the activity of phytanoyl-CoA hydroxylase increased up to four times, depending on the particular cell type. In one cell line, HepG2, no induction of phytanoyl-CoA hydroxylase activity was observed. After addition of phytanic acid to COS-1 cells, an increase in phytanoyl-CoA hydroxylase activity was observed within 2 h, indicating a quick cell response. No stimulation of phytanoyl-CoA hydroxylase was observed when COS-1 cells were grown in the presence of clofibric acid, 9- cis -retinoic acid or both ligands together. This indicates that the activation of phytanoyl-CoA hydroxylase is not regulated via PPAR, or RXR. However, stimulation of PPAR, and all RXRs by clofibric acid and 9- cis -retinoic acid was observed in transient transfection assays. These results suggest that the induction of phytanoyl-CoA hydroxylase by phytanic acid does not proceed via one of the nuclear hormone receptors, RXR or PPAR,. [source] COX-2 inhibits Fas-mediated apoptosis in cholangiocarcinoma cellsHEPATOLOGY, Issue 3 2002Ugochukwu C. Nzeako Fas expression has been shown to negatively regulate the progression of cholangiocarcinoma cells in xenografts. However, many human cholangiocarcinomas express Fas, suggesting these cancers have developed mechanisms to inhibit Fas-mediated apoptosis. Cyclooxygenase-2 (COX-2), which generates prostanoids, is expressed by many cholangiocarcinomas. Therefore, our aim was to determine whether COX-2 expression inhibits death receptor,mediated apoptosis in KMBC cells, a cholangiocarcinoma cell line. These cells express messenger RNA for the death receptors Fas, tumor necrosis factor receptor 1 (TNF-R1), death receptor 4 (DR4), and DR5. Agonists for these death receptors, CH-11, TNF-,, and TRAIL all induced apoptosis. However, COX-2, whether induced by proinflammatory cytokines or transient transfection, only significantly inhibited Fas-mediated apoptosis. The COX-2 inhibitor NS-398 restored Fas-mediated apoptosis in COX-2 transfected cells. Prostaglandin E2 reduced apoptosis and mitochondrial depolarization after treatment with the Fas agonist CH-11. Of a variety of antiapoptotic proteins examined, COX-2/prostaglandin E2 only increased expression of Mcl-1, an antiapoptotic member of the Bcl-2 family. In conclusion, these data suggest that prostanoid generation by COX-2 specifically inhibits Fas-mediated apoptosis, likely by up-regulating Mcl-1 expression. Pharmacologic inhibition of COX-2 may be useful in augmenting Fas-mediated apoptosis of cholangiocarcinoma cells. [source] Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations,HUMAN MUTATION, Issue 9 2010James J. Cox Abstract SCN9Aencodes the voltage-gated sodium channel Nav1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Nav1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Nav1.7-,R1370-L1374). Both of these mutations map to the pore region of the Nav1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Nav1.7. © 2010 Wiley-Liss, Inc. [source] Expression of ,CP-4 inhibits cell cycle progression and suppresses tumorigenicity of lung cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 7 2008Zafira Castaño Abstract The protein ,CP-4 (also known as hnRNP E4) is an RNA binding protein encoded by a gene at 3p21, one of the most common altered regions in lung cancer. It has been proposed that ,CP-4 may function as a lung tumor suppressor. Lack of ,CP-4 expression is frequent in highly proliferative lung tumors and correlates with ,CP-4 allele losses. The aim of this study was to evaluate the effect of ,CP-4 on the tumorigenic capacity of lung cancer cells. ,CP-4 expression was induced by transient transfection or stable infection with recombinant retroviruses. Induction of ,CP-4 expression caused cell cycle arrest in G2/M in 3 out of the 7 lung cancer cell lines studied, while no effect on apoptosis was observed. Anchorage-independent growth and invasion capacity of H1299 cells were significantly reduced by ,CP-4 induction. Tumorigenicity of H1299 cells in nude mice was greatly inhibited by the expression of ,CP-4. Moreover, induction of ,CP-4 expression in already established tumors resulted in a sudden growth arrest. Immunocytochemistry analysis of the xenograft tumors revealed an in vivo effect of ,CP-4 on cell proliferation and no effect on apoptosis. Finally, ,CP-4 showed a subcellular localization different from ,CP-4a, a splice variant that does not affect cell proliferation. In conclusion, expression of ,CP-4 can inhibit proliferation and tumorigenesis of lung cancer cells, both in vivo and in vitro, by delaying the progression of the cell cycle. © 2007 Wiley-Liss, Inc. [source] Direct and indirect manipulation of the MEK-ERK pathway regulates the formation of a pericellular HA-dependent matrix by chick articular surface cells without modifying CD44 expresssionINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004Edward R. Bastow Introduction Recent evidence suggests that hyaluronan (HA) facilitates the mechano-dependent joint cavity-forming process through the elaboration and retention of a HA-rich pericellular matrix in the developing joint interzone (IZ). The presumptive joint IZ phenotype shows a capacity to bind and synthesize HA and also exhibits elevated activated ERK, prior to synovial joint cavity formation (Lamb et al. 2001; Edwards et al. 1994; Dowthwaite et al. 1998). We have found that immobilization, which induces embryonic joint fusion with loss of the joint IZ phenotype, also reduces ERK activity levels in the IZ. As the signalling events regulating the synthesis and binding of HA have yet to be determined, we hypothesize that ERK activation plays a pivotal role in determining the presumptive joint IZ phenotype through HA synthetic and binding capacity. Materials and methods Chick articular surface (AS) cells were harvested from proximal tibiotarsal joints of embryos by collagenase digestion. Pericellular coat formation was assessed using the erythrocyte exclusion assay and cell-coat area ratios determined. ERK activity was modulated by transient transfection of GFP constructs of constitutively active (CA-) or dominant negative (DN-) forms of MEK, the direct upstream regulator of ERK or by treatment with the MEK inhibitor PD98059 (50 µm). ERK activation was monitored by immunochemistry. CD44 expression and ERK activation in PD98059-treated cells were monitored by immunoblotting and medium HA concentrations by ELISA. Results AS cells form large pericellular coats that are lost following hyaluronidase treatment and thus dependent upon HA for their construction. Treatment with PD98059 significantly reduced pericellular coat formation after 6 h. In parallel, we confirmed that PD98059 diminished active ERK expression without modifying overall levels of ERK, suggesting that the elaboration of large HA-pericellular coats is dependent upon MEK's activation of ERK. Western blot analysis of PD98059-treated cells showed that loss of pericellular coats was not, however, associated with any decreased levels of the cell surface HA receptor CD44. Although treatment with PD98059 did not change medium HA concentration after short times of exposure, at times (up to 6 h) during which coat loss was evident, prolonged treatment over 24 h significantly decreased medium HA concentration. Consistent with a role for ERK in pericellular coat formation, transfection with DN-MEK diminished, while CA-MEK increased, both active ERK expression and coat formation efficiency. We also found that, commensurate with this modification in coat forming efficiency, cells expressing DN-MEK exhibited a significant reduction in labelling of free HA on the cell surface. Discussion These studies extend our recent work to indicate that: (i) direct modulation of ERK activation by transfection with its endogenous upstream regulator modifies cell surface-associated HA (ii) PD98059-induced blockade of ERK activation restricts medium HA release and (iii) ERK-mediated changes in pericellular coat elaboration are independent of changes in cellular CD44 expression. These findings suggest an intimate relationship between ERK activation and the formation/retention of HA-rich pericellular matrices in vitro and highlight the role for ERK activation in regulating joint line-related differentiation. [source] TCDD suppresses insulin-responsive glucose transporter (GLUT-4) gene expression through C/EBP nuclear transcription factors in 3T3-L1 adipocytesJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2006Phillip Chin-Chen Liu Abstract TCDD is known to reduce significantly the level of the functionally active form of glucose transporter type 4 (GLUT4) in vivo in adipose tissue and muscles. To study the mechanistic basis of this phenomenon, we conducted transient transfection and DNA deletion analysis in 3T3-L1 cells using chloramphenicol acetyltransferase (CAT) reporter plasmids containing the GLUT4 promoter joined to the bacterial CAT. It was found that in transfected control samples, CAT activity was significantly higher in cells transfected with p469CAT and p273CAT than those with p78CAT, indicating that the region between ,78 and ,273 contained elements that play major roles in transactivation of this gene. Treatment with TCDD decreased CAT activity with p469CAT and p273CAT, but not with p78CAT, indicating the same region to contain the element(s) affected by TCDD. A gel-shift (EMSA) analysis result indicated that TCDD shows the profound effect only on the nuclear proteins binding to the [32P]-labeled probe containing C/EBP response element equivalent of the ,265 to ,242 stretch of the GLUT4 promoter. The results of supershift analysis showed that TCDD caused a decrease in the tier of C/EBP, and an increase in that of C/EBP, among the proteins bound to this C/EBP response element. We studied the effect of TCDD in cells overexpressing either C/EBP,, C/EBP,, or C/EBP, through transient transfection of p273CAT or p469CAT. The results clearly showed that the effect of TCDD to suppress the CAT activity of p273 or p469 disappeared in those cells overexpressing C/EBP, or C/EBP,. These results implicate the C/EBP proteins to be the main mediator of suppressive action of TCDD on GLUT4 gene expression in 3T3-L1 cells. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:79,87, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20120 [source] Two potent transactivation domains in the C-terminal region of human NANOG mediate transcriptional activation in human embryonic carcinoma cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Hyun-Jin Do Abstract The core embryonic stem cell transcription factors Oct4, Sox2, and Nanog are expressed in germ cell tumors (GCTs) and have been proposed to play a regulatory role in tumorigenesis. However, little is known about the mechanism of regulation of tumorigenesis by the complicated network of these proteins. Nanog is a novel homeobox-containing transcription factor that is expressed in pluripotent cells as well as GCTs. To understand the molecular and functional role of human NANOG (hNANOG) in germ cells, mutagenesis of the C-terminal domain (CD) of hNANOG and transient transfection assays in NCCIT human embryonic carcinoma cells were carried out to identify critical transactivation motifs. We divided the CD into three putative functional subdomains, CD1, tryptophan-repeat (WR) subdomain, and CD2. WR subdomain and CD2 independently contained transcriptional potential and, in combination, had a synergistic effect on transcriptional activity, while CD1 was transcriptionally inactive. The glutamine (Q) motif in WR subdomain, and multiple acidic residues in CD2 were required for maximal and synergistic transcriptional activation by the hNANOG CD. The results of the current study contribute to a better understanding of the complicated molecular machinery of stem cell transcription factors and their role in unregulated proliferation in germ cell tumorigenesis. J. Cell. Biochem. 106: 1079,1089, 2009. © 2009 Wiley-Liss, Inc. [source] Inhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: Experiments with a model systemJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Olga N. Umanskaya Abstract We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context. J. Cell. Biochem. 99: 598,608, 2006. © 2006 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||