Regulation of Human Thrombin-Activable Fibrinolysis Inhibitor Gene Expression in Megakaryocyte-Like (Dami) and Monocyte/Macrphage- Like (THP-1) Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3078-3078
Author(s):  
Joellen H. H. Lin ◽  
Michael B. Boffa ◽  
Marlys L. Koschinsky
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Mrna Expression ◽  
Fibrin Clot ◽  
Mrna Abundance ◽  
Luciferase Reporter ◽  
Rt Pcr ◽  
Monocytes And Macrophages ◽  
Fibrinolysis Inhibitor ◽  
Flanking Region

Abstract Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase zymogen defining a pathway that functions as a molecular link between coagulation and fibrinolysis. Activation by thrombin, the thrombin-thrombomodulin complex, or plasmin, the resultant enzyme (TAFIa) affects the balance between these two cascades by attenuating positive feedback in the fibrinolytic cascade, thereby inhibiting fibrin clot lysis. Plasma TAFI antigen levels vary significantly between individuals, which has implicated TAFI as a risk factor for thrombotic diseases. TAFIa can also inactivate pro-inflammatory peptides such as the anaphylatoxins and bradykinin, suggesting a role for the TAFI pathway as a link between coagulation and inflammation. TAFI expression in cultured hepatic cells is decreased by interleukins −1 and −6, and plasma TAFI levels in human are decreased in experimental endotoxemia. Although the liver is the main source of plasma TAFI, TAFI has also been identified in platelets, and TAFI mRNA has been detected in the Dami (megakaryoblastic) cell line (but not the MEG-01 cell line). TAFI mRNA has also been detected in adipocytes of patients with type 2 diabetes; however, TAFI mRNA expression in human umbilical vein endothelial cells is still a point of controversy. It has been hypothesized that platelet TAFI arises from TAFI gene expression in megakaryocytes (MK). Using RT-PCR and real-time RT-PCR, we not only confirmed the presence of TAFI mRNA in Dami cells, but also found that TAFI mRNA abundance was increased throughout Dami cell differentiation along the megakaryocytes/platelet lineage (up to 8 fold increase after 48 hours) stimulated by phorbol myristate acetate (PMA) treatment. The quantitative real-time RT-PCR experiments revealed that TAFI mRNA is present in differentiated Dami cells at a level that is only one-hundredth of that observed in HepG2 (hepatoma) cells. Using transfection experiments with luciferase reporter plasmids containing progressive deletions of the human TAFI 5′-flanking region, we identified the sequence between −438 and −257 (relative to the initiator methionine codon) to be responsible for the enhanced TAFI gene transcription as Dami cells differentiate into more mature MK-like cells. Moreover, using western blot analysis, we detected TAFI protein expression in the medium of differentiated Dami cells, but not untreated Dami cells. Together, these data provide further evidence supporting the idea that platelet TAFI is generated from TAFI gene expression in megakaryocytes rather than by uptake from the plasma. To study TAFI gene regulation in monocytes and macrophages, RT-PCR and realtime RT-PCR were used to detected and quantify, respectively, TAFI mRNA expression in both THP-1 and THP-1 cells that have been differentiated into macrophage-like cells (THP-1ma) by PMA treatment. TAFI mRNA abundance was similar in THP-1 cells as what was observed in differentiated Dami cells. In addition, we found a progressive decrease in TAFI mRNA abundance throughout the THP-1 differentiation with an 85% decrease after 24 hours of PMA treatment. Transfection experiments using luciferase reporter plasmids representing progressive deletions of the human TAFI 5′-flanking region identified sequences between −151 and −121 as harboring key promoter elements for the differentiation-associated decrease in TAFI gene expression as THP-1 differentiate into macrophage-like cells. However, no TAFI protein was detected in either THP-1 or THP-1ma conditioned medium using western blot analyses. Nonetheless, extra-hepatic expression of TAFI, such as platelet, monocytes and macrophages, suggests novel roles for TAFI pathway beyond regulation of fibrin clot breakdown.

Regulation of the Gene Encoding Human Thrombin-Activable Fibrinolysis Inhibitor by Female Sex Steroids

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3077-3077
Author(s):  
Mathieu Garand ◽  
Michael B Boffa ◽  
Marlys L. Koschinsky
Keyword(s):  
Gene Expression ◽  
Sex Steroids ◽  
Promoter Activity ◽  
Mrna Abundance ◽  
Gene Encoding ◽  
Female Sex ◽  
Menopausal Women ◽  
Fibrinolysis Inhibitor ◽  
Flanking Region

Abstract Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme that, once activated by thrombin, the thrombin-thrombomodulin complex, or plasmin, attenuates fibrinolysis. Aberrant regulation of the TAFI pathway alters the balance between coagulation and fibrinolysis and may underlie severe haemostatic disorders such as thrombosis and hemophilia. Indeed, high plasma TAFI levels have been associated with several cardiovascular diseases. It is important to note that there is a large inter-individual variability in plasma TAFI levels within the population, and this variation is primarily due to non-genetic factors. Therefore, variation in TAFI gene expression is a risk factor for thrombotic disorders and may be an important means by which TAFI responds to environmental and physiological stimuli. Novel associations between plasma TAFI levels and sex hormones have triggered interest in determining the role of TAFI as a mediator of the cardioprotective effects of estrogens and progestagens, or as a mediator of the increased thrombotic risk that accompanies use of oral contraceptives or hormone replacement therapy. Plasma TAFI concentrations rise with age in women but not in men, and are elevated in post-menopausal women compared to pre-menopausal women. In addition, plasma TAFI levels have been shown to be decreased by selective estrogen receptor modulators such as HMR 3339 and raloxifene, estradiol plus trimegestone, transdermal estradiol, and oral estradiol plus gestodene. On the other hand, some studies have reported minimal to no change in plasma TAFI levels occurring with the use of oral contraceptive, Raloxifene, or Tamoxifen. Paradoxically, it has been shown that both plasma TAFI levels and clot lysis time rise during pregnancy and then promptly return to basal levels after delivery. These studies illustrate the controversies surrounding the role of sex steroids in modulating plasma TAFI levels. In the present study, we have attempted to directly measure the effect of sex steroids on hepatic TAFI gene expression, and to uncover the molecular mechanisms underlying these regulatory events. HepG2 (human hepatocellular carcinoma) cells were cultured in the presence or absence of progesterone and b-estradiol and TAFI mRNA abundance was measured using real-time RT-PCR. We found that both of these hormones significantly decrease endogenous TAFI mRNA abundance in a dose-dependant manner. To assess the ability of these hormones to influence transcription of the gene encoding TAFI, we treated HepG2 cells that had been transiently transfected with luciferase reporter plasmids containing the 5′-flanking region of the TAFI gene. Interestingly, the change in promoter activity closely paralleled changes in mRNA abundance, suggesting that the effect of the hormones is mediated at the level of transcription. Furthermore, changes in TAFI mRNA abundance following treatments with estrogen were not associated with a decrease in TAFI mRNA stability when compared to the untreated control. TAFI protein levels were also decreased in a dose-dependent manner as assessed by western blot analysis. Inspection of the sequence of the TAFI 5′-flanking region does not show any consensus estrogen responsive elements, although we cannot exclude a role for more complex transcriptional system such as an estrogen response unit. The effect of estrogen could also be performed indirectly through the modulation of other transcription factors such SP-1 or members of the basal transcriptional machinery. We also investigated whether progesterone decreases TAFI gene expression via the binding of the progesterone receptor to the established glucocorticoid responsive element (GRE) within the TAFI promoter. Our results showed that progesterone generates the same decrease in promoter activity even when the GRE site was mutated, indicating that progesterone may act through a different site. In conclusion, our studies are beginning to reveal the molecular basis for the apparent relationship between female sex steroids and plasma concentrations of TAFI: specifically, a direct downregulatory effect on transcription of the gene encoding TAFI.

Global expression analysis of ECL cells in Mastomys natalensis gastric mucosa identifies alterations in the AP-1 pathway induced by gastrin-mediated transformation

2004 ◽  
Vol 20 (1) ◽  
pp. 131-142 ◽  
Author(s):  
M. Kidd ◽  
T. Hinoue ◽  
G. Eick ◽  
K. D. Lye ◽  
S. M. Mane ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gastric Mucosa ◽  
Western Blot ◽  
Cell Transformation ◽  
Mastomys Natalensis ◽  
Rt Pcr ◽  
Cell Hyperplasia ◽  
Ecl Cells ◽  
Ecl Cell ◽  
Gene Expression Alterations

Enterochromaffin-like (ECL) cell hyperplasia and then irreversible neoplasia can be generated in the African rodent Mastomys natalensis using the H2 receptor blocker, loxtidine, for 8–16 wk. We used a GeneChip approach complemented by standard technologies to identify gene expression alterations in the gastric mucosa during gastrin-mediated ECL cell transformation. Gastric mucosa (mucosal scrapping) and ECL cell-enriched fractions were obtained from untreated Mastomys (controls) and from animals treated with loxtidine for 8 wk (hyperplasia). Tumor ECL cells were obtained by hand-dissection of gastric ECL cell nodules from animals treated with loxtidine for >16 wk and from a spontaneously developed ECL cell tumor. RNA was isolated, examined on rat U34A GeneChips, and comparison analysis was performed to identify altered gene expression. Alterations in gene expressions were examined further by immunohistochemistry, quantitative RT-PCR (Q-RT-PCR), sequencing and Western blot. GeneSpring analysis demonstrated alterations in few genes (<20) in hyperplastic and tumor mucosa. The histamine H1 receptor was consistently increased in proliferating mucosa. This gene change was confirmed by Q-RT-PCR. Other genes showing alterations included neural-(chromogranin A and somatostatin), cell-cycle-, and AP-1-associated genes. Immunostaining confirmed alterations in neural markers. Cluster analysis of ECL cell-enriched samples demonstrated that c- fos and junD were differently regulated. Q-RT-PCR and Western blot in prospectively collected gastric mucosal samples confirmed the differential expression of Fos and Jun. The negative regulators of AP-1, JunD, and Menin were decreased in tumor mucosa. A missense of unknown function was noted in the menin gene. Hypergastrinemia in an animal model of gastric carcinoids differentially altered the histamine type 1 receptor and gene expression and protein composition of AP-1. These results suggest that expression of this receptor and an altered composition of AP-1 with a loss of inhibition play a role in ECL cell transformation.

scholarly journals Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells

2000 ◽  
Vol 348 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Isabelle VAN SEUNINGEN ◽  
Michaël PERRAIS ◽  
Pascal PIGNY ◽  
Nicole PORCHET ◽  
Jean-Pierre AUBERT
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Start Site ◽  
Transcription Start ◽  
Mucin Gene ◽  
Intron 1 ◽  
Flanking Region

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5ʹ-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5ʹ-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor ĸB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2α and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

scholarly journals Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 331-340
Author(s):  
WE Kaminski ◽  
E Jendraschak ◽  
K Baumann ◽  
R Kiefl ◽  
S Fischer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Time Course ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Constitutive Expression ◽  
Ionophore A23187 ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Human Mononuclear Cells

Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

scholarly journals Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 331-340 ◽  
Author(s):  
WE Kaminski ◽  
E Jendraschak ◽  
K Baumann ◽  
R Kiefl ◽  
S Fischer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Time Course ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Constitutive Expression ◽  
Ionophore A23187 ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Human Mononuclear Cells

Abstract Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

Proteomic Signature Predicting Achievement of Very Good Partial Response In Patients with Multiple Myeloma Based On Complementary Label-Free and iTRAQ Quantitative Proteome Analysis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1902-1902
Author(s):  
Dominik Dytfeld ◽  
Malathi Kandarpa ◽  
John R Strahler ◽  
Dattatreya Mellacheruvu ◽  
Suchitra Subramani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Western Blot ◽  
Differential Expression ◽  
Proteomic Analysis ◽  
Quantitative Proteomics ◽  
Proteomic Profile ◽  
Differentially Expressed Proteins ◽  
Label Free ◽  
Rt Pcr

Abstract Abstract 1902 Introduction: Multiple myeloma (MM) remains mostly incurable. Novel therapies have improved response rates, which are now reaching 100%. More importantly, number of recent studies showed that the depth of response, e.g. achievement of at least 90% reduction of the disease (≥VGPR) is associated with longer disease control. Therefore, improving VGPR rates and establishing predictors of VGPR to a given regimen may be an important clinical goal. High throughput quantitative proteomics may offer greater insight into the actual biology of the malignant cell than genome analysis and therefore, may be more useful in the development of personalized therapy. The objective of this study is to establish a proteomic signature predicting achievement of at least VGPR to initial treatment with bortezomib (Velcade®), pegylated liposomal doxorubicin, and dexamethasone (VDD). We previously reported preliminary proteomic profile of malignant plasma cells (PCs) obtained from a set of naïve MM pts enrolled in the VDD trial (Dytfeld et al., ASH 2009). Here we present the results of differential proteomic analysis of MM PCs of all available samples from the frontline VDD study (≥VGPR vs. <VGPR) using two independent and complementary quantitative proteomic platforms. We also compared the proteomic profile with gene expression data. Preliminary validation of the biomarkers of response prediction is presented. Methods: PCs were acquired from pre-treatment bone marrow specimens after obtaining informed consent from patients (pts), and were thereafter enriched with a RosetteSep® negative selection kit. Quantitative proteomic analysis of PCs from 17 naïve pts with MM from the VDD study was performed using iTRAQ approach in 8-plex variant. To increase confidence of analysis, label-free quantitative proteomics (LF) based on spectra counting was conducted on PCs from 12 pts. In iTRAQ experiments, proteins were processed with reagents according to the manufacturer's protocol followed by SCX fractionation and LC-MS/MS analysis (4800 Plus MALDI TOF/TOF). Peptides from the MM1S cell line were used as a reference. The data were analyzed using ProteinPilot™. For LF analysis, proteins were fractionated before trypsin digestion on Bis-Tris-Gel and subsequently run on LC-ESI-MS/MS on a linear trap mass spectrometer (LTQ Orbitrap). A database search was carried out using X!Tandem followed by Trans-proteomic Pipeline. At least 1.5-fold difference in expression in both platforms was used as a cut-off value. To correlate proteomics with gene expression of dysregulated proteins of interest, mRNA levels were analyzed by quantitative real time PCR (RT-PCR). Validation of proteomic findings on proteins of interest was performed using Western Blot. Results: We identified a total of 894 proteins in 3 iTRAQ experiments with high confidence (FDR<1%) and 1058 proteins by LF approach. Based on iTRAQ analysis, 20 proteins were found up-regulated in samples from pts with ≥VGPR (8 out of 17 pts) while 14 were down- regulated. Using LF approach, 284 proteins were elevated in the ≥VGPR group (6 out of 12 pts) while 315 proteins were down-regulated. Both iTRAQ and LF methods showed 15 differentially expressed proteins in common and 14 of them showed identical up or down trends. Interestingly, among differentially expressed proteins, there were proteins involved in proteasome activation (PSME1 and TXNL1), protection against oxidative stress (TXN and TXNDC5), glucose and cholesterol metabolism (TP1, APOA1 and ACAT1) and apoptosis (MX1). RT-PCR performed on a subset of genes confirmed the trend in differential expression between pts with ≥VGPR and <VGPR for TXNDC5 and PSME1. No change in mRNA expression levels was observed in TXN, APOA1, TPI1 and MX1while the trend in expression was reversed for ACAT1. Western blot analysis performed to date validated differential expression of PSME1. Conclusions: We present patient-derived proteomic characteristics of MM cells using two independent proteomic platforms. As a proof of concept, analysis of PCs obtained from pts enrolled in the frontline VDD study shows differential expression of 34 proteins in pts who achieved ≥VGPR vs. pts with <VGPR. Correlation with gene expression and further validation and functional analysis are in progress. This study was supported by a grant from the Multiple Myeloma Research Foundation. Disclosures: Jakubowiak: Millennium, Celgene, Bristol-Myers Squibb, Johnson & Johnson Ortho-Centocor: Honoraria; Millennium, Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millennium, Celgene, Centocor-Ortho Biotech: Speakers Bureau.

scholarly journals Oestrogen receptor α is required for biochanin A-induced apolipoprotein A-1 mRNA expression in HepG2 cells

2007 ◽  
Vol 98 (3) ◽  
pp. 534-539 ◽  
Author(s):  
Ming Yan Chan ◽  
Gho Wai Man ◽  
Zhen-yu Chen ◽  
Jun Wang ◽  
Lai K. Leung
Keyword(s):  
Mrna Expression ◽  
Hepg2 Cells ◽  
Oestrogen Receptor ◽  
Transcriptional Control ◽  
Plasma Lipid ◽  
Firefly Luciferase ◽  
Mrna Abundance ◽  
Biochanin A ◽  
Luciferase Reporter ◽  
Postmenopausal Symptoms

Epidemiological studies have indicated that soya consumption may produce a better plasma lipid profile. The effect may be attributed to the phyto-oestrogens in soya. The red clover (Trifolium pratense) isoflavone biochanin A has a chemical structure similar to those phyto-oestrogens found in soya beans, and is marketed as a nutraceutical for alleviating postmenopausal symptoms. In the present study we investigated the effect of biochanin A on the mRNA expression of ApoA-1 in the hepatic cell line HepG2. Real-time PCR revealed that biochanin A increased ApoA-1 mRNA abundance in cells expressing oestrogen receptor (ER) α. Without ERα transfection, biochanin A had no effect on mRNA abundance. In order to study the transcriptional control, a fragment of the 5′-flanking region of the ApoA-1 gene was amplified and inserted in a firefly luciferase reporter plasmid. The reporter assay indicated that the transactivation of the ApoA-1 promoter was induced by biochanin A in HepG2 cells transfected with the ERα expression plasmid. This induction was reduced by the anti-oestrogen ICI 182,780, whereas the inhibitors of protein kinase (PK) C, PKA, or mitogen-activated kinase (ERK) had no suppressive effect. The present study illustrated that biochanin A might up regulate hepatic apoA-1 mRNA expression through an ER-dependent pathway.

scholarly journals Identification and characterization of a silencer regulatory element in the 3′-flanking region of the murine CD46 gene

2000 ◽  
Vol 351 (2) ◽  
pp. 353-365 ◽  
Author(s):  
Midori NOMURA ◽  
Akira TSUJIMURA ◽  
Nasim A. BEGUM ◽  
Misako MATSUMOTO ◽  
Hiroetsu WABIKO ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Stop Codon ◽  
Regulatory Element ◽  
Luciferase Reporter ◽  
Size Difference ◽  
Binding Factor ◽  
Gene Assay ◽  
Flanking Region ◽  
Silencer Element

The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3´-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein–silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46.

Overexpression of the Anti-Apoptotic Genes mcl-1, bcl-w, bcl-xL and a1 Is Correlated with Chronic Myelogenous Leukemia Progression and Resistance to Gleevec.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2880-2880
Author(s):  
F. A. Castro ◽  
J. F. Jacysyn ◽  
A. G. Ulbrich ◽  
P. R. Tobo ◽  
L. R. Lopes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Real Time ◽  
Chronic Phase ◽  
Rt Pcr ◽  
Blastic Crisis ◽  
Gapdh Gene ◽  
Blastic Phase ◽  
Cytogenetic Remission ◽  
Apoptotic Genes

Abstract Chronic myeloid leukemia (CML) is a stem cell disease where t(9;22) translocation is considered the primary molecular event leading to the appearance of the bcr-abl fusion gene and consequent cellular transformation. Bcr-Abl tyrosine-kinase inhibitors have been developed and are fairly successful in the treatment of CML. Despite their outstanding clinical activity in CML, they are not a definitive cure: the efficacy of imatinib mesylate (Gleevec®), for instance, in CML-blastic phase is reduced and reports of resistance and intolerance to it have been published. Since Bcr-abl initiates cellular modifications leading to an extreme resistance to apoptosis, we decided to investigate possible secondary targets for CML therapy. We evaluate the expression of known anti and pro-apoptotic genes in terms of CML progression and response to Gleevec. We studied 10 health controls and 71 CML patients in different phases (20 chronic phase, 20 accelerated phase, 10 blastic phase, 15 cytogenetic remission post-Gleevec® and 6 Gleevec® refractory patients). CML group was constituted by 26 men and 35 women, median age of 51.7 years (range 23–73 years), 5 men and 5 women, median age of 49.3 (range 25–72 years), were healthy controls. Peripheral blood mononuclear cells were isolated and expression of bax, bcl-w, mcl-1, bcl-2, a1 and bcl-xL was analyzed by real-time RT-PCR. Protein expression of Bcl-2 and Bcl-xL were analyzed by western-blot. The results of real-time RT-PCR and western-blot are expressed by relative expression, e.g. ratio of investigated genes or protein to the reference GAPDH gene and protein, respectively. We observed an increase of bcl-w (p<0.001), mcl-1 (p< 0.001), a1 (p<0.01) and bcl-xL (p<0.001) gene expression and a remarkable reduction of bcl-2 (p<0.001) in CML-BP patients (table 1). Patients in remission post-Gleevec® presented an anti-apoptotic gene expression profile similar to controls (p>0.05) and refractory patients profile seem to be analogous to blastic crisis (p>0.05). bax levels did not show significant changes in CML patients in different phases (p>0.05). Bcl-2 and Bcl-xL protein data support real-time RT-PCR findings. Taken together these results suggest that mcl-1, bcl-w, bcl-xL and a1 contribute to disease progression and resistance to treatment in CML patients. Further investigations on the state of the apototic machinery in CML patients should provide new approaches for drug design and consequently new efficient treatment for AP, BC and refractory CML patients. Table 1. Ratio of amplicons of the investigated genes to housekeeping (GAPDH). Gene C CP-CML AP-CML BP-CML CCR-CML R-CML C: control; CP: chronic phase; AP: accelerated phase; BP: blastic crisis; CCR: complete cytogenetic remission; R: refractory patients. Results expressed by mean /SD. mcl-1 3.6/0.9 4.4/1.0 8.5/5.0 12.6/2.7 2.4/0.7 13.7/3.6 a1 329.9/153.3 564.1/349.1 1,7000/564.4 972.4/564.0 434.2/98.1 968.8/2.4 bcl-w 4.2/1.1 12.8/3.2 3.4/0.6 17.2/8.6 3.5/0.8 19.8/3.2 bcl-xL 2.9/1.3 5.4/1.1 10.6/3.5 39.4/6.2 3.0/2.1 42.0/3.5 bcl-2 21.1/5.2 13.8/7.0 6.1/3.1 3.1/1.3 21.7/6.4 3.6/2.2 bax 3.2/1.3 4.2/0.8 3.7/1.3 5.2/2.5 4.5/2.7 4.2/2.7

Defibrotide Counteracts the Modifications of Anti-Thrombotic Phenotype of Endothelial Cells Induced by Thalidomide.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2502-2502 ◽  
Author(s):  
Cinara Echart ◽  
Barbara Graziadio ◽  
Cinzia Repice ◽  
Mario Boccadoro ◽  
Antonio Palumbo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Fibrin Clot ◽  
Rt Pcr ◽  
Plasminogen Activator Inhibitor 1 ◽  
Adhesive Properties ◽  
Pai 1

Abstract Introduction: Patients with Multiple myeloma are at relatively high risk of developing thromboembolic events, usually deep vein thromboses (DVT). There are numerous contributing factors, including therapy, such as thalidomide, where DVT has been identified as a major toxicity, especially when thalidomide is used in combination with other treatments such as dexamethasone. The mechanisms by which thalidomide predisposes to thrombosis are not well understood. Defibrotide (DF) is an orally biovailable polydisperse oligonucleotide with anti-thrombotic, pro-fibrinolytic and anti-adhesive properties. Previously, DF has been shown to dose-dependently counteracted the increase in Plasminogen Activator Inhibitor-1 (PAI-1) expression and decrease on tissue plasminogen activator (t-PA) activity after lipopolysaccharide (LPS) stimulation of endothelial cells in vitro. Methods and Results: We have conducted in vitro studies using human microvascular endothelial cells (HMEC) in order to investigate the effect of different doses of thalidomide on various fibrinolytic factors. In addition, we evaluated whether DF modulates changes of fibrinolysis induced by thalidomide. HMEC were treated with 50 and 100μg/ml of thalidomide for 24 hours in presence and absence of DF (at a dose of 150μg/ml). t-PA and PAI-1 gene expression were evaluated through real time polymerase chain reaction (RT-PCR) of cDNA prepared from HMEC. Release of t-PA and PAI-1 were evaluated by imunoenzymatic assay (ELISA). Furthermore, we evaluated the fibrinolytic activity of cell surpernatant using a fibrin clot plate assay. In this method the fibrin clot was formed by mixing fibrinogen, plasminogen and thrombin. The plasmin generated by the cell surpernatant was able to digest fibrin and also hydrolyzed the chromogenic substrate S-2251. The RT-PCR results showed that thalidomide reduces t-PA (2.2 fold) and increases PAI-1 gene expression (4.0 fold) in HMEC cells, whereas DF was able to counteract this effect by up-regulating the t-PA and down-regulating PAI-1 gene expression induced by thalidomide (8.8 and 2.0 fold, respectivielly). Similar results was observed analyzing t-PA release by HMEC cells treated with different concentrations of thalidomide with and without DF. Thalidomide significantly reduces the t-PA released in both concentrations (p&lt;0.001) and DF significantly increase the release of t-PA reduced by thalidomide (p&lt;0.01). The changes of fibrinolytic activity in HMEC by thalidomide and the capacity of DF to restore the fibrinolysis was confirmed by analyzing the lyses of fibrin clots with endothelial cell surpernatant (p&lt;0.01). Conclusions: These results show that DF is able to counteract the alterations of fibrinolytic factors in HMEC treated with thalidomide. Whilst further studies in preclinical MM models are underway, these data suggest a potential role for DF in the prevention of DVT induced by thalidomide and support ongoing clinical trials of DF in combination with thalidomide-based treatment.

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