Tf Gene Expression Research Articles

Decreasing SIL1-R2 concentration could enhance the expression oftranscription factors on the gene level

By the time you opened this document, an individual somewhere in the world has developed Alzheimer’s disease. With over 6 million cases in the United States alone and a global total exceeding 50 million, Alzheimer’s contributes to around one-third of senior fatalities. Horrifyingly, according to the United Nations, the number of patients doubles every two decades if a breakthrough is not discovered. Therefore, it is imperative to the survival of the human species to address and attempt to solve this issue before it can escalate any further. In this work, I have designed a few experiments that could be monumental to developing future Alzheimer’s vaccines and medicine. The primary focus of the trials above is to explore the possible role of the IL-1 signaling protein in the Bace-1- 1 deletion process to the potential prevention of DAM-2 formation, which, in turn, could worsen Alzheimer’s symptoms. The overall prediction can be summed up in the following: The activation of the IL-1 signaling pathway will enhance the activity of TF and lead to the up-regulation of TF gene expression. If my assumption is proven correct, it could pave the way for future Alzheimer’s medication to include drugs that moderate IL-1 signaling, which could save countless amount of lives.

Pycallingcards: an integrated environment for visualizing, analyzing, and interpreting Calling Cards data.

Unraveling the transcriptional programs that control how cells divide, differentiate, and respond to their environments requires a precise understanding of transcription factors' (TFs) DNA-binding activities. Calling cards (CC) technology uses transposons to capture transient TF binding events at one instant in time and then read them out at a later time. This methodology can also be used to simultaneously measure TF binding and mRNA expression from single-cell CC and to record and integrate TF binding events across time in any cell type of interest without the need for purification. Despite these advantages, there has been a lack of dedicated bioinformatics tools for the detailed analysis of CC data. We introduce Pycallingcards, a comprehensive Python module specifically designed for the analysis of single-cell and bulk CC data across multiple species. Pycallingcards introduces two innovative peak callers, CCcaller and MACCs, enhancing the accuracy and speed of pinpointing TF binding sites from CC data. Pycallingcards offers a fully integrated environment for data visualization, motif finding, and comparative analysis with RNA-seq and ChIP-seq datasets. To illustrate its practical application, we have reanalyzed previously published mouse cortex and glioblastoma datasets. This analysis revealed novel cell-type-specific binding sites and potential sex-linked TF regulators, furthering our understanding of TF binding and gene expression relationships. Thus, Pycallingcards, with its user-friendly design and seamless interface with the Python data science ecosystem, stands as a critical tool for advancing the analysis of TF functions via CC data. Pycallingcards can be accessed on the GitHub repository: https://github.com/The-Mitra-Lab/pycallingcards.

Canonical BAF complex activity shapes the enhancer landscape that licenses CD8+ T cell effector and memory fates

CD8+ Tcells provide host protection against pathogens by differentiating into distinct effector and memory cell subsets, but how chromatin is site-specifically remodeled during their differentiation is unclear. Due to its critical role in regulating chromatin and enhancer accessibility through its nucleosome remodeling activities, we investigated the role of the canonical BAF (cBAF) chromatin remodeling complex in antiviral CD8+ Tcells during infection. ARID1A, a subunit of cBAF, was recruited early after activation and established de novo open chromatin regions (OCRs) at enhancers. Arid1a deficiency impaired the opening of thousands of activation-induced enhancers, leading to loss of TF binding, dysregulated proliferation and gene expression, and failure to undergo terminal effector differentiation. Although Arid1a was dispensable for circulating memory cell formation, tissue-resident memory (Trm) formation was strongly impaired. Thus, cBAF governs the enhancer landscape of activated CD8+ Tcells that orchestrates TF recruitment and activity and the acquisition of specific effector and memory differentiation states.

A modERN resource: identification of Drosophila transcription factor candidate target genes using RNAi.

Transcription factors (TFs) play a key role in development and in cellular responses to the environment by activating or repressing the transcription of target genes in precise spatial and temporal patterns. In order to develop a catalog of target genes of Drosophila melanogaster TFs, the modERN consortium systematically knocked down the expression of TFs using RNAi in whole embryos followed by RNA-seq. We generated data for 45 TFs which have 18 different DNA-binding domains and are expressed in 15 of the 16 organ systems. The range of inactivation of the targeted TFs by RNAi ranged from log2fold change -3.52 to +0.49. The TFs also showed remarkable heterogeneity in the numbers of candidate target genes identified, with some generating thousands of candidates and others only tens. We present detailed analysis from five experiments, including those for three TFs that have been the focus of previous functional studies (ERR, sens, and zfh2) and two previously uncharacterized TFs (sens-2 and CG32006), as well as short vignettes for selected additional experiments to illustrate the utility of this resource. The RNA-seq datasets are available through the ENCODE DCC (http://encodeproject.org) and the Sequence Read Archive (SRA). TF and target gene expression patterns can be found here: https://insitu.fruitfly.org. These studies provide data that facilitate scientific inquiries into the functions of individual TFs in key developmental, metabolic, defensive, and homeostatic regulatory pathways, as well as provide a broader perspective on how individual TFs work together in local networks during embryogenesis.

Colchicine inhibits the prothrombotic effects of oxLDL in human endothelial cells

BackgroundTissue Factor (TF) plays a pivotal role in coronary thrombosis. Oxidized low-density lipoproteins (oxLDL) are crucial in development of atherosclerosclerosis. Moreover, oxLDL are known to induce TF expression on several cell types including endothelial cells. The lectin-type oxidized LDL receptor 1 (LOX-1) represent the oxLDL receptor. Colchicine is an anti-mitotic drug recently proven to have beneficial effects in cardiovascular disease via unknown mechanisms. Thus, we aim at investigating colchicine effects on TF expression in oxLDL stimulated human vascular endothelial cells (HUVEC). Some molecular mechanism(s) potentially involved were investigated. MethodsHUVEC were pre-incubated with colchicine 10 μM for 1 h and then stimulated with oxLDL (50 μg/mL). TF gene (RT-PCR), protein (western blot), surface expression (FACS) and procoagulant activity (FXa generation assay) were measured. TF translocation to cell surface was investigated by immunofluorescence. NF-κB/IκB axis was examined by western blot analysis and translocation assay. Finally, LOX-1 expression was also investigated. ResultsColchicine significantly reduced TF gene and protein expression as well as its procoagulant activity in oxLDL-treated HUVEC. These effects seem to be related mainly to action of colchicine on microtubules that, in turn, modulate TF trafficking in the cytoplasm, NF-κB/IκB pathway and LOX-1 expression. ConclusionsData of the present study, although in vitro, indicate that one of the hypothetical mechanisms by which colchicine exert protective cardiovascular effects might be its ability to inhibit the pro-thrombotic activity of oxLDL.

Procoagulant Microparticles Derived from Myeloma Plasma Cells Have a Determinant Role in the Hypercoagulable State Associated with Multiple Myeloma. a Modelization in Vitro Study

Background: Multiple myeloma (MM) is a plasma cell malignancy characterized by bone marrow infiltration leading to multiple lytic bone lesions, renal failure, anemia and increased rate of thrombotic events. The cross-talk between cancer cells, plasma clotting mechanism, platelets and endothelial cells enhances hypercoagulability. The mechanisms by which myeloma plasma cells (MPCs) interfere with their microenvironment during the blood coagulation process have been poorly investigated. Several studies have established a link between levels of cancer cell derived procoagulant microparticles, cancer aggressiveness, response to anticancer treatments well as with the risk of cancer associated thrombosis., The mechanisms by which myeloma plasma cells interfere to blood coagulation process in their microenvironment have been poorly investigated. Aim: Set up an experimental model that allows the identification of the procoagulant fingerprint of MPCs and myeloma cell-derived microparticles (MC-dMPs) as well as their impact on thrombin generation of normal plasma. In addition, we evaluated the relative impact of TF-pathway and the intrinsic clotting pathway on thrombin generation triggered by myeloma plasma cells and MC-dMPs. Finally, we performed a simulation of thrombin generation process in the presence of myeloma plasma cells and MC-dMPs as well as in the presence of varying concentrations of TF and procoagulant phopsholipids, mimicking some of the conditions that potentially exist in MPCs. Methods. TF and annexin V expression by MPCs (MPC-dMPs) were analyzed by flow cytometry, TF activity (TFa) and TF gene expression was also determined. Thrombin Generation (TG) in the presence of MPCs or MPC-dMPs was assessed with the Calibrated Automated Thrombogram assay (CAT®) in normal human PPP. TG was also assessed in plasma spiked with MPCs and MPC-dMPs or variable concentrations of TF and procoagulant phospholipids. Results: MPC-dMPs expressed about 8-fold higher levels of TF as compared to MPCs. TFa expressed by MPC- dMPs was significantly higher as compared to that expressed by MPC cells. MPCs and PC- dMPs enhanced thrombin generation of human plasma. Thrombin generation was significantly higher with MPC-dMPs compared to MPCs. MPCs and MPC-dMPs, when concomitantly present in normal PPP, significantly amplified thrombin generation as compared to that observed in the presence of MPCs alone. Presence of TF and procoagulant phospholipids in the microenvironment resulted in significant amplification of thrombin generation induced by MPCs. Conclusion: This study support the concept that hypercoagulability induced by myeloma plasma cells is the result of the combination of the procoagulant properties of myeloma cells with the procoagulant elements of the plasma microenvironment which consist of procoagulant phospholipids and low TF concentration. Following this concept we assume that MPC-dMPs released by myeloma plasma cells into the microenvironment enhance the weak procoagulant activity of myeloma plasma cells. Whereas this combined effect of MPCs and MPC-dMPs on thrombin generation has an impact on the formation of local fibrin network their critical role into the hypercoagulablity, procoagulant MC-dMP could be a potential target for the evaluation of the aggressivity of MPCs and of their response to anti-myeloma treatment. Disclosures Dimopoulos: Sanofi Oncology: Research Funding. Mohty:Jazz Pharmaceuticals: Honoraria, Research Funding. Elalamy:Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; LEO Pharma: Honoraria, Research Funding; Aspen: Honoraria, Research Funding; Bayer Healthcare: Honoraria, Research Funding.

Transcription Factors Regulation in Human Peripheral White Blood Cells during Hypobaric Hypoxia Exposure: an in-vivo experimental study

High altitude is a natural laboratory, within which the clinical study of human physiological response to hypobaric hypoxia (HH) is possible. Failure in the response results in progressive hypoxemia, inflammation and increased tissue oxidative stress (OxS). Thus, investigating temporal changes in key transcription factors (TFs) HIF-1α, HIF-2α, NF-κB and NRF2 mRNA levels, relative to OxS and inflammatory markers, may reveal molecular targets which contrast deleterious effects of hypoxia. Biological samples and clinical data from 15 healthy participants were collected at baseline and after rapid, passive ascent to 3830 m (24 h and 72 h). Gene expression was assessed by qPCR and ROS generation was determined by EPR spectroscopy. Oxidative damage and cytokine levels were estimated by immuno or enzymatic methods. Hypoxia transiently enhanced HIF-1α mRNA levels over time reaching a peak after 24 h. Whereas, HIF-2α and NRF2 mRNA levels increased over time. In contrast, the NF-κB mRNA levels remained unchanged. Plasma levels of IL-1β and IL-6 also remained within normal ranges. ROS production rate and markers of OxS damage were significantly increased over time. The analysis of TF-gene expression suggests that HIF-1α is a lead TF during sub-acute HH exposure. The prolongation of the HH exposure led to a switch between HIF-1α and HIF-2α/NRF2, suggesting the activation of new pathways. These results provide new insights regarding the temporal regulation of TFs, inflammatory state, and ROS homeostasis involved in human hypoxic response, potentially also relevant to the mediation of diseases that induce a hypoxic state.

Information-dense transcription factor binding site clusters identify target genes with similar tissue-wide expression profiles and serve as a buffer against mutations

Background: The distribution and composition of cis-regulatory modules (e.g. transcription factor binding site (TFBS) clusters) in promoters substantially determine gene expression patterns and TF targets, whose expression levels are significantly regulated by TF binding. TF knockdown experiments have revealed correlations between TF binding profiles and gene expression levels. We present a general framework capable of predicting genes with similar tissue-wide expression patterns from activated or repressed TF targets using machine learning to combine TF binding and epigenetic features. Methods: Genes with correlated expression patterns across 53 tissues were identified according to their Bray-Curtis similarity. DNase I-accessible promoter intervals of direct TF target genes were scanned with previously derived information theory-based position weight matrices (iPWMs) of 82 TFs. Features from information density-based TFBS clusters were used to predict target genes with machine learning classifiers. The accuracy, specificity and sensitivity of the classifiers were determined for different feature sets. Mutations in TFBSs were also introduced to in silico examine their impact on cluster densities and the regulatory states of target genes. Results: We initially chose the glucocorticoid receptor gene (NR3C1), whose regulation has been extensively studied, to test this approach. SLC25A32 and TANK were found to exhibit the most similar expression patterns to this gene across 53 tissues. A Decision Tree classifier exhibited the largest area (0.9987 and 0.9956 respectively before and after eliminating inaccessible promoter intervals based on DNase I HyperSensitive Regions (DHSs)) under the Receiver Operating Characteristic (ROC) curve in detecting such coordinately regulated genes. Target gene prediction was confirmed using siRNA knockdown data of TFs, which was more accurate than CRISPR inactivation. In-silico mutation analyses of TFBSs also revealed that one or more information-dense TFBS clusters in promoters are required for accurate target gene prediction. Conclusions: Machine learning based on TFBS information density, organization, and chromatin accessibility accurately identifies gene targets with comparable tissue-wide expression patterns. Multiple, information-dense TFBS clusters in promoters appear to protect promoters from the effects of deleterious binding site mutations in a single TFBS that would effectively alter the expression state of these genes.

An In Vivo Study on Intoxicating Effects of Nerium oleander Water Based Extract on Multiorgans of Wistar Rat.

This study was aimed to find histological changes in the extrahepatic organs, hepatic iron deposition, and gene expression of some iron regulatory proteins in rats after sterile muscle abscess during the acute intoxication of Nerium oleander leaves decoction. 10 ml/kg of the leaves extract was injected intramuscularly in Wistar rats (200–225 g, n = 4). Control animals received saline injection of matched volume. Animals were anesthetized and sacrificed after 3, 6, 12, and 24 h after administration of decoction. Lungs, kidney, spleen, and liver were extracted and processed for histopathological examination while portion of liver tissue was proceeded for iron regulatory gene expression quantification. Sections of all studied organs were found with signs of cellular dysfunction with infiltration of variety of leucocytes. In the lungs section at 3 h time point mononuclear cell infiltrates were observed while in alveolar tissue at 24 h time point dilation and even collapse in some of the alveoli were evident. In kidney sections distortion of renal tubules and epithelial cells with shrinkage of glomeruli was noted at all studied time points. In the splenic section of 12 h time point, degeneration, depopulation, and shrinkage of white pulp have been noted. Distension of the red pulp along with activation of splenic follicles was evident after 24 h onset of APR. Significant changes in the expression of acute phase cytokine and iron regulatory genes were noted. IL-6 and Hepc gene expression were strongly upregulated up to 12 h whereby Tf gene expression showed an early upregulation at 3 h time point followed by downregulation on later points while Hjv gene expression showed an overall downregulation at all study time points compared to control. It is concluded that inherent toxins present in the N. oleander can induce acute phase response and cause severe histological changes in the organs and marked changes in the regulation of iron regulatory proteins thus cannot be practiced routinely.

Fine-tuned iron availability is essential to achieve optimal adipocyte differentiation and mitochondrial biogenesis.

Adipose tissue from obese and insulin-resistant individuals showed altered expression of several iron-related genes in a recent study, suggesting that iron might have an important role in adipogenesis. To investigate this possible role, we aimed to characterise the effects of iron on adipocyte differentiation. Intracellular iron deficiency was achieved using two independent approaches: deferoxamine administration (20 and 100 μmol/l) and transferrin knockdown (TF KD). The effects of added FeSO4, holo-transferrin and palmitate were studied during human and 3T3-L1 adipocyte differentiation. Finally, the relationship between iron-related and mitochondrial-related genes was investigated in human adipose tissue. Most adipose tissue iron-related genes were predominantly expressed in adipocytes compared with stromal vascular cells. Of note, transferrin gene and protein expression increased significantly during adipocyte differentiation. Both deferoxamine and TF KD severely blunted adipocyte differentiation in parallel with increased inflammatory mRNAs. These effects were reversed in a dose-dependent manner after iron supplementation. Palmitate administration also led to a state of functional intracellular iron deficiency, with decreased Tf gene expression and iron uptake during adipocyte differentiation, which was reversed with transferrin co-treatment. On the other hand, iron in excess impaired differentiation, but this antiadipogenic effect was less pronounced than under iron chelation. Of interest, expression of several genes involved in mitochondrial biogenesis occurred in parallel with expression of iron-related genes both during adipogenesis and in human adipose tissue. Precise and fine-tuned iron availability is essential to achieve optimal adipocyte differentiation, possibly modulating adipocyte mitochondrial biogenesis.

Anatomical and transcriptional dynamics of maize embryonic leaves during seed germination

Our anatomical analysis revealed that a dry maize seed contains four to five embryonic leaves at different developmental stages. Rudimentary kranz structure (KS) is apparent in the first leaf with a substantial density, but its density decreases toward younger leaves. Upon imbibition, leaf expansion occurs rapidly with new KSs initiated from the palisade-like ground meristem cells in the middle of the leaf. In parallel to the anatomical analysis, we obtained the time course transcriptomes for the embryonic leaves in dry and imbibed seeds every 6 h up to hour 72. Over this time course, the embryonic leaves exhibit transcripts of 30,255 genes at a level that can be regarded as "expressed." In dry seeds, ∼25,500 genes are expressed, showing functional enrichment in transcription, RNA processing, protein synthesis, primary metabolic pathways, and calcium transport. During the 72-h time course, ∼13,900 genes, including 590 transcription factor genes, are differentially expressed. Indeed, by 30 h postimbibition, ∼2,200 genes expressed in dry seeds are already down-regulated, and ∼2,000 are up-regulated. Moreover, the top 1% expressed genes at 54 h or later are very different from those before 30 h, reflecting important developmental and physiological transitions. Interestingly, clusters of genes involved in hormone metabolism, signaling, and responses are differentially expressed at various time points and TF gene expression is also modular and stage specific. Our dataset provides an opportunity for hypothesizing the timing of regulatory actions, particularly in the context of KS development.

Opposite regulation by PI3K/Akt and MAPK/ERK pathways of tissue factor expression, cell-associated procoagulant activity and invasiveness in MDA-MB-231 cells

BackgroundTissue factor (TF), an initiator of blood coagulation, participates in cancer progression and metastasis. We recently found that inhibition of MAPK/ERK upregulated both full length TF (flTF) and soluble isoform TF (asTF) gene expression and cell-associated TF activity in breast cancer MDA-MB-231 cells. We explored the possible mechanisms, especially the possible interaction with EGFR and PI3K/Akt pathways.MethodsA plasmid containing TF promoter −2174 ~ +128 plus luciferase reporter gene was introduced into MDA-MB-231 cells to evaluate TF promoter activity. In order to study the interaction of these pathways, ERK inhibitor (PD98059), PI3K inhibitors (LY294002, wortmannin), Akt inhibitor (A6730), and EGFR inhibitor (erlotinib) as well as the corresponding siRNAs were used to treat MDA-MB-231 cells, and ovarian cancer OVCAR-3 and SKOV-3 cells. Quantitative PCR and western blot were used to determine TF expression. One stage clotting assays were used to measure pro-coagulation activity of the MDA-MB-231 cells.ResultsWe show that PI3K inhibitors LY294002, wortmannin and A6730 significantly inhibited TF promoter activity, and reduced TF mRNA and protein levels due to the inhibition of Akt phosphorylation. In contrast, ERK inhibitor PD98059 and ERK siRNA enhanced TF promoter activity by 2.5 fold and induced an increase in TF mRNA and protein levels in a dose dependent manner in these cells. The PI3K/Akt pathway was shown to be involved in PD98059-induced TF expression because the induction was inhibited by PI3K/Akt inhibitors. Most interestingly, the EGFR inhibitor erlotinib and EGFR siRNA also significantly suppressed PD98059- or ERK siRNA-induced TF promoter activity and TF protein expression. Similar results were found with ovarian cancer cells SKOV-3 and OVCAR-3. Furthermore, in MDA-MB-231, mRNA levels of asTF were regulated in a similar way to that of TF in response to the cell treatment.ConclusionsThis study showed a regulatory mechanism in which MAPK/ERK signals inhibit EGFR/PI3K/Akt-mediated TF expression in breast cancer MDA-MB-231 cells. The same regulation was observed in ovarian cancer OVCAR-3 and SKOV-3 cells. Interestingly, we observed that both flTF and asTF could be regulated in a parallel manner in MDA-MB-231. As the PI3K/Akt pathway and EGFR regulate TF expression in cancer cells, targeting these signaling components is expected to potentially inhibit TF expression-associated tumor progression.

A viral histone H4 suppresses expression of a transferrin that plays a role in the immune response of the diamondback moth, Plutella xylostella

A transferrin (Tf) gene has been predicted from an expressed sequence tag of the diamondback moth, Plutella xylostella. It encodes 681 amino acid residues that share 80-90% sequence homologies with other lepidopteran Tfs. The gene was constitutively expressed in all developmental stages of P. xylostella. Double-stranded RNA (dsRNA) specific to the Tf gene was prepared and microinjected into the larvae. We hypothesize that the dsRNA treatment suppressed the Tf gene expression level and it significantly inhibited haemocyte nodule formation in response to bacterial challenge. The larvae treated with dsRNA also showed a significantly enhanced susceptibility to an entomopathogenic bacterium, Bacillus thuringiensis. An endoparasitoid wasp, Cotesia plutellae, parasitized the larvae of P. xylostella, which showed significant reduction of Tf expression. The suppression of Tf expression was mimicked by transient expression of a viral gene CpBV-H4, encoded in the symbiotic virus of C. plutellae. A truncated form of CpBV-H4 prepared by deleting an extended N-terminal 38 amino acid residue lost its inhibitory activity against the Tf gene expression. These results suggest that Tf of P. xylostella plays an immunological role in P. xylostella and that the suppression of its expression in the parasitized larvae is caused by a viral histone H4 in an epigenetic mode.

Tumor Expression of Alternative Spliced Tissue Factor Is a Prognostic Marker in Non-Small Cell Lung Cancer.

Abstract 335▪▪This icon denotes an abstract that is clinically relevant. Introduction.Tissue Factor (TF), a 47-kDa transmembrane protein, is the essential receptor for factor VII/VIIa and the physiological trigger of blood coagulation. TF is directly involved in the evolution of several cancers by promoting tumor growth, angiogenesis, cell migration and development of metastases. We recently demonstrated that high tumor expression of TF was associated with reduced survival in non-small cell lung cancer (NSCLC). In addition, combined oncogene events affecting K-RAS, TP53, PTEN and LKB1 genes were shown to dramatically increase TF gene expression in lung tumors (Regina et al., Clin Chem 2009, in press). Recently, the presence of an alternatively spliced TF isoform (asTF) has also been evidenced in human blood and various normal or cancerous tissues. This soluble isoform is lacking exon 5 that encodes the transmembrane domain, and several studies suggested that it could also be involved in angiogenesis and tumor progression. Aim of the study.To evaluate the level of asTF transcripts in NSCLC tumors and to look for a relationship with biological and clinical features and particularly TF and VEGF gene expression, and the survival of affected patients. Methods.The expression of the asTF transcript was assessed by a real-time PCR method based on Taqman strategy within the lung tumors of 57 patients with NSCLC. Results were normalized according to 18S RNA expression by calculating the 2-Dct = 2-(ctasTF - ct18S) for each sample and were compared to full-length TF (flTF) mRNA levels and to VEGF165 or VEGF189 gene expression. Levels of asTF transcripts were also analyzed according to the status, i.e. wild type or mutated, of K-RAS, TP53, PTEN and LKB1 within tumors. Results.asTF mRNA was detectable in all samples, but the level of transcripts was highly variable from one tumor to another (median 5.32×10-5; range 1.16×10-7-2×10-2). No association could be found between the level of asTF transcripts and those of flTF. In addition, the expression of asTF in lung tumors did not correlate with VEGF165 and VEGF189 mRNA levels and was similar in tissue samples without and with mutations of K-RAS, TP53, PTEN or LKB1. When levels of asTF transcripts were analyzed according to clinico-pathological features, no association with sex, age, stage, differentiation grade, TNM classification or histological type, was found. In contrast, Kaplan-Meier curves analysis showed that patients with high asTF tumor expression (above the median value) had a poorer prognosis with reduced survival than patients with tumors in which lower levels of asTF transcripts had been measured (HR 3.1; CI 95%: 1.2-6.5, p=0.01, Log rank test). Moreover, analysis of data using Cox's regression model showed that asTF tumor expression is an independent prognosis marker in NSCLC (RR = 3.08; CI 95%: 1.35-7.01, p = 0.0076). Importantly, the best outcome was evidenced in patients for whom tumor levels of both asTF and flTF transcripts were low. The survival was therefore significantly reduced when either asTF or flTF expression in tumors was high while the poorest prognosis was recorded when tissue levels of both isoforms were high (p=0.018, Log rank test). Discussion-Conclusion.Our study demonstrates that tumor expression of alternative spliced TF is an independent prognostic marker in patients with non-small cell lung cancer. How asTF does influence the progression of cancer remains unknown, although its role in angiogenesis has been recently suggested. However, no association between tumor levels of asTF transcripts and VEGF165/VEGF189 gene expression could be evidenced in our study. Mechanisms that regulate TF isoform expression in tumor cells are also poorly identified. We previously showed that tumor expression of flTF is associated in NSCLC to the accumulation of mutations of several genes involved in the mTOR pathway. Our present data support that these mutations do not influence the alternative splicing of TF in lung tumors, which could be specifically regulated by the PI3K/Akt pathway as recently demonstrated in human endothelial cells (Einsereich et al, Circ J 2009, in press). Disclosures:No relevant conflicts of interest to declare.

Genoproteomic Mining of Urothelial Cancer Suggests γ-Glutamyl Hydrolase and Diazepam-Binding Inhibitor as Putative Urinary Markers of Outcome after Chemotherapy

Urinary biomarkers for the detection of bladder cancer have been developed, but no similar markers exist for prediction of clinical outcomes after receiving chemotherapy. Here we evaluate an approach that combines genomic, proteomic, and therapeutic outcome datasets to identify novel putative urinary biomarkers of clinical outcome after neoadjuvant methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC). Using this method, we identified gamma-glutamyl hydrolase (GGH), emmprin, survivin, and diazepam-binding inhibitor (DBI). Interestingly, GGH is a protein associated with methotrexate resistance, whereas emmprin, survivin, and DBI had been previously identified as predictors of outcome after platinum-containing chemotherapeutic regimens when assessed on tumor tissue. Using disease-free survival as a marker for clinical outcome, we evaluated the ability of GGH, emmprin, survivin, and DBI expression in tumor tissue to stratify 27 patients treated with neoadjuvant MVAC. DBI (P = 0.046) but not GGH (P = 0.190), emmprin (P = 0.066), or survivin (P = 0.393) successfully stratified patients. When GGH was used with DBI the significance of stratification improved (P = 0.024), whereas the addition of survivin or emmprin to this latter two-gene model reduced its significance (P = 0.036 and P = 0.040, respectively). Although these predictive results were obtained on tumor tissues, the presence of GGH and DBI in urine serves as a rationale for developing them as urinary markers of clinical outcomes for patients treated with neoadjuvant MVAC.

Hypoxia-inducible Factor-1 Deficiency Results in Dysregulated Erythropoiesis Signaling and Iron Homeostasis in Mouse Development

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of genes whose products play critical roles in energy metabolism, erythropoiesis, angiogenesis, and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif1alpha gene, which encodes the hypoxia-responsive alpha subunit of HIF-1, causes cardiac, vascular, and neural malformations resulting in lethality by embryonic day 10.5 (E10.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-1alpha-deficient embryos, as well as decreased hemoglobin content in erythroid colonies from HIF-1alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif1alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif1alpha-/- embryos. The erythropoietic defects in HIF-1alpha-deficient erythroid colonies could not be corrected by cytokines, such as vascular endothelial growth factor and Epo, but were ameliorated by Fe-SIH, a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis, Hif1alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin, Fpn1, Irp1, and frascati. We conclude that dysregulated expression of genes encoding Epo, EpoR, and iron regulatory proteins contributes to defective erythropoiesis in Hif1alpha-/- yolk sacs. These results identify a novel role for HIF-1 in the regulation of iron homeostasis and reveal unexpected regulatory differences in Epo/EpoR signaling in yolk sac and embryonic erythropoiesis.

The Shear Stress Response Element of Endothelium and the expression of TF Gene(Cardiovascular Mechanics)

The Shear Stress Response Element of Endothelium and the expression of TF Gene(Cardiovascular Mechanics)

Estrogen regulation of transferrin gene expression in MCF-7 human breast cancer cells.

Transferrin (Tf) is an iron transport protein expressed in MCF-7 human breast cancer cells. In nuclear run-on assays, 17beta-estradiol (E2) increased the rate of Tf gene expression approximately 3-fold within 1 h after treatment and reporter gene activity was also induced in MCF-7 cells transfected with a construct containing a -3600 to +39 Tf gene promoter insert. Deletion and mutation analysis identified an E2-responsive promoter region between -811 and -762, which was GC-rich (80%) and contained two nonconsensus estrogen response elements (EREs). E2-responsiveness of this region was associated with a GGACA(N)(3)TGGCC motif (-803 to -791) which bound human estrogen receptor alpha (hERalpha) in gel mobility shift assays. In Drosophila Schneider SL-2 cells, the -811 to -752 was E2-responsive after cotransfection with hERalpha expression plasmid plus E2, whereas Sp1 protein did not induce transactivation. These studies confirm that E2 induces Tf gene expression through a nonconsensus distal ERE.

Mechanism of Resveratrol-mediated Suppression of Tissue Factor Gene Expression

Tissue factor (TF) is a cell surface receptor for factor VII(a), and the binding of factor VII(a) to TF initiates the coagulation cascade. Inappropriate in vivo expression of TF in vascular cells has been shown to be responsible for thrombotic disorders associated with a variety of pathological conditions, including gram-negative sepsis, cancer and atherosclerosis. A number of epidemiological studies suggest that moderate consumption of red wine provides protective effects against coronary heart disease mortality. Recently, we have shown that resveratrol, a polyphenolic compound found in wine, inhibited the induction of TF expression in endothelial cells and mononuclear cells (Pendurthi UR, Williams JT, Rao LVM. Arterioscler Thromb Vasc Biol 1999: 19: 419-426). In the present study, we examined the mechanism by which resveratrol inhibits the expression of TF in monocytes by using a monocytic cell line, THP-1, as a model cell. Northern blot analysis, gel mobility shift assays and transfection studies with various TF promoter constructs, as well as other transcription regulatory constructs, were used to elucidate the inhibitory mechanism of resveratrol. The data show that resveratrol inhibited lipopolysaccharide (LPS)-induced expression of TF in human monocytes and monocytic cell line, THP-1 in a dose dependent manner. Resveratrol did not significantly alter the binding of various transcription factors involved in TF gene expression to DNA. However, resveratrol suppressed the transcription of cloned human TF promoter. Further experiments revealed that resveratrol reduced kappaB- but not AP-1-driven transcriptional activity. Additional experiments showed that resveratrol suppressed the phosphorylation of p65 and its transactivation. In summary, our results indicate that resveratrol does not inhibit the activation or translocation of NF-kappaB/Rel proteins but inhibits NF-kappaB/Rel-dependent transcription by impairing the transactivation potential of p65.

Opposite regulation of tissue factor expression by calcineurin in monocytes and endothelial cells.

Tissue factor (TF), the primary initiator of blood coagulation with structural homology to the cytokine receptor family, has been implicated in various vascular processes including metastasis, angiogenesis, and atherosclerosis. Within the vasculature, monocytes and endothelial cells (EC) can be activated to synthesize TF depending on the induction of NF-kappaB. Despite the undisputed value of cyclosporin A (CsA) as an immunosuppressant, problems have emerged due to induction of vascular changes by a poorly understood mechanism. We demonstrate that CsA has opposite effects on TF gene expression, inhibiting NF-kappaB-mediated TF gene transcription in monocytes but enhancing it in EC. To test whether CsA binding proteins (cyclophilins) can mediate these CsA effects we used a nonimmunosuppressant analog of CsA that binds to cyclophilins but does not inhibit the Ca2+/calmodulin-dependent phosphatase calcineurin (Cn). This drug lacked regulatory function for NF-kappaB and TF expression suggesting that Cn is responsible for the inverse gene regulation. The key function of Cn was supported by experiments demonstrating that other phosphatase inhibitors also either positively or negatively regulated NF-kappaB in monocytes and EC. Calcineurin was demonstrated to regulate NF-kappaB activation at the level of IkappaBalpha degradation, because agonist-induced phosphorylation and subsequent degradation of IkappaBalpha is prevented by Cn inhibitors in monocytes but enhanced in EC. These data identify Cn as an opposite regulator in generating transcriptionally active NF-kappaB, and they confirm the presumption that the ability of Cn to participate in NF-kappaB transactivation is not T cell specific.