Dissertations / Theses on the topic 'NF-kB activity'

In the last decade there has been a thriving development of quantitative methods in the field of biomedical physics, both in the modeling of biological complex systems and in the statistical analysis of experimental results. In this thesis we examine the effect of a variety of stimuli on the NF-kB activity's oscillation in metastatic colorectal cancer cells using probabilistic models such as the Chemical Master Equation and the Bayesian statistics. After a brief introduction we explain the theoretical foundations of the Master Equation approach and of the Bayesian inference, together with a brief dip in the subject of Hamiltonian Monte Carlo methods. In the continuation we create a solvable model using the Master Equation approach and we gain insights concerning its eigenvalues distribution in the complex plane. We therefore build a Bayesian regression model and we use it to analyze the oscillating autocorrelation function of the previous stochastic model in order to test the capabilities of the statistical model. Finally we analyze the biological data using the previously created and tested statistical model, exploring and commenting the results and outlining further research directions.

Stimulation of Toll-like receptors (TLRs) in cells of the innate immune system activates the expression of a proinflammatory and antimicrobial gene program controlled by a network of transcriptional regulators. We show that NFAT5, which belongs to the Rel family of transcription factors and was previously characterized as an osmostress responsive factor, is required for the expression of a group of TLR-responsive genes in macrophages, such as Nos2, Il6 and Tnf. NFAT5 recruitment to its target genes is dependent on IKKβ activity, de novo protein synthesis and is sensitive to histone deacetylases. Interestingly, NFAT5 is essential in the response to low doses of TLR ligands, regulating specific gene subsets depending on the stimulus strength. We also show that macrophages use NFAT5 to facilitate chromatin accessibility, allowing the recruitment of transcriptional regulators such as p65/NF-κB, c-Fos and p300 to its target regions. We use Nos2 as a gene whose induction is NFAT5-dependent especially at low doses of LPS to demonstrate that NFAT5 controls the recruitment of p65 by facilitating the activity of H3K27 demethylases, without influencing the binding of Polycomb repressive complex 2 or JMJD3. Altogether, this thesis characterizes NFAT5 as a novel regulator of the immune response to low pathogen load involved in the control of local chromatin accessibility.
En las células del sistema inmunitario innato, la estimulación de los receptores de tipo Toll (TLR) activa la expresión de un programa génico pro-inflamatorio y antimicrobiano que está controlado por una red de reguladores transcripcionales. Hemos demostrado que el NFAT5, perteneciente a la familia de factores de transcripción Rel y previamente caracterizado como un factor de respuesta a estrés osmótico, es importante para la expresión de un grupo de genes de respuesta a TLRs, entre ellos Nos2, Il6 y Tnf. El reclutamiento del NFAT5 a sus genes diana requiere la actividad de IKKβ, la síntesis de novo de proteínas y es sensible a la acción de las deacetilasas de histonas. Resulta interesante el hecho de que el NFAT5 es esencial para responder a bajas dosis de ligando de los TLRs, y que regula grupos de genes específicos dependiendo de la intensidad del estímulo. También mostramos que NFAT5 facilita la accesibilidad de la cromatina en macrófagos, permitiendo el reclutamiento de reguladores transcripcionales como p65/NF-kB, c-Fos y p300 a sus regiones diana. Utilizando Nos2 como un gen cuya inducción es más dependiente de NFAT5 a bajas dosis de LPS, demostramos que el NFAT5 controla el reclutamiento de p65 gracias a que facilita la actividad de las demetilasas de H3K27, pero sin influir en la unión del complejo Polycomb 2 ni JMJD3. En conclusión, esta tesis caracteriza al NFAT5 como un nuevo regulador del sistema inmunitario implicado en el control de la accesibilidad local de la cromatina en respuesta a baja carga de patógenos.

La maggior parte dei tumori riescono ad evadere il sistema immunitario inibendo l’espressione di antigeni tumorali associati alle molecole del complesso maggiore di istocompatibilità di classe I (MHC I) sulla superficie cellulare. La mancata espressione di questi complessi è spesso dovuta alla presenza di difetti strutturali dei geni codificanti le molecole MHC I, oppure all’aberrante espressione delle molecole responsabili del processamento degli antigeni legati alle molecole MHC I. Il neuroblastoma (NB), il tumore extracraniale solido più comune dell’infanzia, non è un’eccezione. Sia la maggior parte delle linee cellulari di NB, che i tumori primari esprimono bassi, se non nulli, livelli di MHC I che possono essere aumentati trattando le cellule con l’interferone-gamma (IFN-γ). Questo fenotipo è compatibile con la presenza di difetti nella regolazione trascrizionale delle molecole coinvolte nel processamento e nella presentazione dell’antigene. Il presente studio ha lo scopo di indagare il meccanismo molecolare che determina la mancata o ridotta espressione delle molecole MHC I, e delle due aminopeptidasi del reticolo endoplasmatico ERAP1 ed ERAP2. Le forme più aggressive di NB sono caratterizzate dall’amplificazione dell’oncogene MYCN. Sebbene una correlazione inversa tra l’espressione di MYCN le molecole MHC I nelle linee cellulari umane di NB sia stata riportata, un coinvolgimento diretto di MYCN nella regolazione di MHC I non è stato dimostrato. I nostri risultati dimostrano che MYCN non è responsabile dei bassi livelli di MHC I, ERAP1 ed ERAP2 nelle cellule di NB analizzate, infatti la loro espressione non è influenzata nè dalla forzata espressione ne dall’inibizione di MYCN. Abbiamo invece identificato due fattori di trascrizione, NF-kB e IRF1, che sono direttamente coinvolti nella regolazione delle proteine MHC I ed ERAPs. Mediante il saggio di immunoprecipitazione della cromatina abbiamo dimostrato che il reclutamento di p65 (una subunità di NF-kB) sui promotori di MHC I, ERAP1 ed ERAP2 è direttamente proporzionale all’espressione di questi geni. Inoltre, il fenotipo negativo per MHC I, ERAP1 ed ERAP2, caratteristico delle forme più aggressive di NB, coincide con una bassa attività nucleare di NF-kB ed IRF1. L’overespressione dei due fattori trascrizionali da soli è in grado di recuperare solo parzialmente l’espressione di MHC I, ERAP1 ed ERAP2, inoltre il risultato dipende dalla linea cellulare trasfettata. Comunque, la trasfezione contemporanea di NF-kB ed IRF1 produce un incremento sinergico dei geni target in tutte le linee trasfettate. Degno di nota è il fatto che l’espressione di p65 nei tumori primari di neuroblastoma è simile a quella osservata nelle linee cellulari. Infatti, solo le cellule gangliari, ovvero le cellule più differenziate presenti nel tessuto tumorale, esprimono sia l’MHC I che il p65 nucleare. Quindi, questo studio mette in luce il meccanismo molecolare responsabile della mancata espressione delle molecole MHC I, ERAP1 ed ERAP2 nei tumori di neuroblastoma più aggressivi fornendo un importante punto di partenza per lo sviluppo di protocolli immunoterapeutici più efficaci basati sull’utilizzo delle cellule T.
Low expression of major histocompatibility complex class I (MHC I) molecules on the cell surface allows tumors to evade the host T cell-based immune response. These abnormalities are often related to either genetic defects of MHC I genes or aberrant expression of antigen processing machinery (APM) components. Neuroblastoma (NB), the most common solid extracranial cancer of childhood, is not an exception. MHC I surface expression is virtually undetectable in the most NB cell lines and primary tumors, and upregulated by gamma-interferon (IFN-γ). This phenotype is compatible with defects in the regulation of antigen processing and presentation components. In this study, the molecular mechanism underlying low immunogenicity in neuroblastoma was investigated. Amplification of the MYCN oncogene characterizes the most aggressive forms of NB and is believe to downregulate expression of MHC class I molecules. Although an inverse correlation between MYCN and MHC I has been reported in human NB cell lines, a direct demonstration of the MYCN-mediated down-regulation of MHC I expression has been questioned. Herein, we demonstrate that MYCN is not responsible for low MHC I, ERAP1 and ERAP2 protein levels in human NB cell lines, since their expression is not affected by neither transfection-mediated overexpression nor siRNA suppression of MYCN. Instead, we identified NF-kB and IRF1 as the main factors involved in the transcriptional regulation of MHC I and ERAPs proteins. By chromatin immunoprecipitation assay, we show a recruitment of p65 NF-kB to the MHC I, ERAP1 and ERAP2 promoters that is proportional with the expression of these genes. Moreover, low nuclear activity of both NF-kB and IRF1 factors correlated with the MHC I, ERAP1 and ERAP2-low phenotype of the most aggressive NB cell lines. Overexpression of either the transcription factors alone rescued the MHC I, ERAP1 and ERAP2-low phenotype, but only partially and in a cell-type depending manner. Important, the co-transfection of both NF-kB and IRF1 cooperated to strongly enhance the transactivation of MHC I, ERAP1 and ERAP2 in any cell lines. Notheworthy, NF-kB and IRF1 acted in a synergistic manner. We found an intriguing parallel in primary NB tumors, in fact, nuclear p65 was detected in the maturing neuroblastic cells (i.e. ganglionic cells) which express higher levels of MHC I molecules in human NB specimens. These findings provide molecular insight into defective MHC I expression in NB tumors and indicate that activating NF-kB and IRF1 in MHC I-low, aggressive NB cells could be instrumental for successful application of T cell-based immunotherapy.

Thesis (Ph. D.)--West Virginia University, 2001.
Title from document title page. Document formatted into pages; contains xi, 147 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 128-143).

The tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were initially discovered as proteins that inducibly interact with the intracellular region of TNF receptors (TNFRs). Because the TNFRs lack intrinsic catalytic activity, the TRAFs are hypothesized to orchestrate signaling activation downstream of the TNFR superfamily, however their mechanism of activation remains unclear (Inoue et al., 2000; Bishop, 2004). Originally, the TRAFs were compared to the signal transducers and activators of transcription (STAT) protein family, due to their sequence homology, and the presence of multiple RING- and zinc-finger domains, suggesting that their function may be to regulate transcriptional activity (Rothe et al., 1994; Hu et al., 1994; Sato et al. 1995; Cheng et al., 1995). However, subsequent research focused predominantly on their cytoplasmic functions, and more recently on their function as E3 ubiquitin ligases (Pineda et al., 2007). In my research, I analyzed the subcellular localizations of the TRAFs following CD40 ligand (CD40L)-stimulation, and found that TRAF2 and 3 rapidly translocate into the nucleus of primary neurons and Neuro2a cells. Interestingly, similar analysis conducted in pre-B lymphocytes (Daudi cells) revealed a different response to CD40L-stimulation, with TRAF2 and 3 being rapidly degraded within 5-minutes of stimulation. These findings are significant because they demonstrate for the first time that the TRAFs translocate into the nucleus and suggest that they may function within the nucleus in a cell-specific manner. I next analyzed the ability of TRAF2 and 3 to bind to DNA, and found that they both bind to chromatin and the NF-kappaB consensus element in Neuro2a cells, following CD40L-stimulation. Similar analyses of the chromatin binding of TRAF2 and 3 in Daudi cells revealed that they were rapidly degraded, similar to the results from my analysis of their subcellular localization. These findings show for the first time that the TRAFs interact with DNA, and therefore support the hypothesis that the TRAFs may function within the nucleus as transcriptional regulators. Finally, I analyzed the ability of the TRAFs to regulate transcriptional activity by luciferase assay. Previous studies showed that overexpression of TRAF2 and 6 could induce NF-kappaB transcriptional activity; however researchers have not been able to determine the mechanism by which they do so. In my studies, I found that every TRAF can directly regulate transcriptional activity either as co-activators or co-repressors of transcription, in a cell- and target protein-specific manner. Additionally, I found that TRAF2 can act as a transcriptional activator, and that its ability to regulate transcription is largely dependent upon the presence of its RING-finger domain. In conclusion, these studies have revealed an entirely novel function for the TRAFs as immediate-early transcriptional regulators. Future research into the genes that are regulated by the specific TRAF complexes will further elucidate how the TRAFs regulate TNFR signaling, as well as whether dysfunctions in TRAF signaling may be associated with known disorders. If specific TRAF complexes are found to regulate specific genes, then pharmacological targeting of the individual TRAF complexes may allow for the highly specific inhibition of signaling events downstream of the TNFRs, without compromising overall receptor signaling, transcription factor pathways, or cellular systems.

INTRODUCTION AGEs and ALEs (Advanced Glycoxidation/Lipoxidation End products) are covalently modified proteins that can act as pathogenic factors in several chronic diseases, like diabetes and cardiovascular diseases. These covalent adducts are formed by different mechanisms. AGEs are proteins covalently modified by reducing sugars or their oxidative degradation products, involving the Maillard reaction. ALEs are proteins modified by reactive carbonyl species (RCS) generated by lipid peroxidation. AGEs/ALEs can be the basis of many different pathologies, underlining the importance for good analytical methods for identification and characterization for the use of biomarkers, but also as a drug target. However, the identification, characterization and quantification of AGEs/ALEs remains to be very challenging due to heterogeneous precursors (sugars, lipids) leading to heterogeneous AGEs/ALEs, present in low concentrations and being very complex analytes. Various techniques to identify and characterize AGEs/ALEs have been described, making use of an isolation/enrichment step based on reactive groups, like carbonyls. However, not all AGEs/ALEs retain reactive groups and therefore can not be isolated and identified using these techniques, indicating the need for a new strategy. The strategy that has been employed in our laboratory is to use the soluble domain of the RAGE receptor, VC1, to affinity enrich AGEs. Using this approach, AGEs/ALEs will be enriched independently of the protein and type of modification. Moreover, a ligand of RAGE can be identified, which could be a potential biomarker of a disease caused by oxidative stress. RAGE is a type I cell surface receptor that is expressed in several cells, such as endothelial cells, smooth muscle cells, but also dendritic cells and T-lymphocytes and is predominantly located in the lungs. The receptor has been implicated in many different pathologies with a marked oxidative base, such as diabetes, atherosclerosis and neurodegenerative diseases. One of the pathways that can be activated is the Nf-κB pathway. The Nf-κB pathway is the ideal signaling pathway to investigate the binding and activation of RAGE by AGEs or ALEs. For this purpose, a cell line was obtained with and without overexpression of RAGE. Furthermore, the cell lines were transfected with a Nf-κB reporter gene, providing us with a fast and high-throughput assay for the evaluation of a pro-inflammatory response upon stimulation with AGEs/ALEs. AIM OF THE PROJECT The identification and characterization of AGEs/ALEs has proven to be crucial in the onset and development of many pathologies. Therefore, good analytical strategies need to be developed/optimized for better understanding of the exact nature of modification, to understand the role they play in disease progression. Identified AGEs/ALEs can serve as biomarker, as well as drug targets. The VC1 technique was proven to be a promising technique to accommodate the need for enrichment of AGEs for better characterization. The first aim of the project was therefore to investigate whether also ALEs are binder of RAGE, since they share the same structural properties than AGEs, and also have been shown to activate the Nf-κB pathway, implicating a role for receptors, like RAGE. Furthermore, to gain a deeper insight into the molecular mechanisms involved in the protein-protein engagement. Since a successful enrichment strategy was developed, the second aim of this project was focused on identifying AGEs/ALEs in biological samples. The first part was focused on oxidizing healthy human plasma in-vitro using AAPH as a radical initiator, and the incubation of plasma directly with RCS, anticipating the production of AGEs/ALEs. The VC1 technique was then used to identify which AGEs/ALEs are produced. Simultaneously, other variables during the sample preparation and analysis were optimized. As explained before, AGEs/ALEs are present in very low concentrations in biological samples, hence the need for very sensitive methods and instrumentation allowing identification. Since human serum albumin (HSA) is the main protein present in plasma, around 50-60%, and has multiple nucleophilic targets, it represents the best model for characterizing AGEs/ALEs. For this reason, the focus was on extracting HSA from plasma, using the newest generation of tribrid MS for the analysis of AGEs/ALEs in plasma samples. AGEs are ligands for RAGE, meaning, they can bind and activate the receptor, inducing a signaling pathway and pro-inflammatory response. ALEs have also been shown to induce a pro-inflammatory response; however, no specific receptor has been linked to this cellular event. Using a cell line with and without RAGE, we aimed to determine whether ALEs can bind and activate the Nf-κB pathway through RAGE. RESULTS AND DISCUSSION ALEs as binder of RAGE In order to investigate the interaction between RAGE and ALEs, different ALEs were produced in-vitro by incubating HSA with different concentrations of well-known lipid derived RCS and in particular: ACR, MDA and HNE. After 24, 48 and 72 h, aliquots of the incubation mixtures were withdrawn, and the reaction was stopped by removing the excess of RCS by ultrafiltration. Intact protein analysis by direct infusion MS was used to evaluate the extent of HSA modifications and demonstrated that by using a wide range of molar ratios and different time-points a quite wide array of ALEs for each tested RCS was generated. In order to characterize ALEs selectively enriched by RAGE, a VC1 pull-down assay was performed as previously described. HSA and HSA treated with MDA, ACR or HNE were assayed for binding to VC1-resins and to control resin. As expected, unmodified HSA was not retained by the VC1-resin. At increasing molar ratios and incubation time, higher amounts of albumin modified with MDA or ACR were eluted from the VC1 resin, with a predominance of the high molecular weight (HMW) species. The modified albumins were retained by the VC1-resin, but not by the control resin. ALEs in the reaction mixtures and those enriched by VC1 were analyzed by bottom-up MS in order to identify the PTMs and to localize the amino acid residues involved in the protein adduct formation. With regard to MDA, only di-hydropyridine adducts on lysines (DHPK), and N-2-pyrimidyl-ornithine adducts on arginines (NPO) were retained by VC1-domain. The n-propenal modifications of lysine (NPK), largely identified before enrichment, were not identified after the enrichment. ACR induced a set of modifications which were identified only after VC1 enrichment and in particular the N-(3-formyl-3,4-dehydro-piperidinyl) lysine (FDPK) modifications, the Michael adduct on cysteines, the double Michael adduct of lysines, the Michael adduct of histidine, the N-2-(4 hydroxy-tetrahydro-pyrimidyl) ornitine (propane-arginine, HTPO) and the Nε-(3-methylpyridinium)-lysine (MP-lysine). Most of the ALEs generated by HNE were found both before or after enrichment, with the exception of a few Michael adducts which were selectively retained by VC1 (not detected before enrichment). With a view to rationalizing the key factors influencing the RAGE binding of the monitored adducts, in silico studies were performed. They were focused on the adducts on arginine and lysine residues as formed by ACR and MDA since they are numerous, with a very broad range of affinity, thus allowing the development of clear structure-affinity relationships. RAGE-ligand interacting regions are characterized by a rich set of positively charged residues which can bind acidic regions of a protein. The mechanism identified using in silico studies, involves a basic amino acid at the center of carboxylic acids like glutamate and aspartate, which forms a set of ionic bridges. Once the basic amino acid is modified by ACR or MDA to an adduct with a neutral charge, the carboxylic acids become available to freely contact the RAGE positive residues. Identification of AGEs/ALEs in biological samples The VC1 technique has proven to be successful in enriching AGEs and ALEs, so the next step was to exploit this technique in biological samples. In order to identify proteins prone to be modified due to oxidative pathways, and possibly serve as biomarker, healthy human plasma was oxidized using the radical initiator AAPH. Different concentrations of AAPH and different timepoints were tested for the presence of protein carbonyl groups, an indicator for protein oxidation and possibly the formation of AGEs/ALEs. A time and concentration dependent formation of carbonyl groups is observed in plasma. Next, samples were analyzed using a bottom-up approach. Results obtained were showing many oxidation products, such as amino side chain oxidation, however no AGEs/ALEs were identified. Thus, a new approach was adopted, including the incubation of plasma directly with RCS, such as HNE, MDA and ACR. This resulted in the formation of AGEs/ALEs in plasma samples, however, they could not be retained by the VC1 domain. Instead of using the VC1 technique to enrich AGEs/ALEs from biological samples, other variables throughout the experimental set-up were optimized. Previously, peptides were analyzed using the Orbitrap LTQ XL, a very powerful instrument. Nonetheless, the newest generation of tribrid MS offers even higher resolution, and it increases protein coverage due to parallel isolation and detection, and faster analyzers. Moreover, we focused on AGEs/ALEs from HSA and using NaBH4 to reduce and stabilize adducts throughout the analysis. This new approach permitted us to identify many AGEs/ALEs in both healthy human plasma samples, but also AGEs/ALEs only present in heart failure samples. Glycation on lysine residues was the main modification identified, present in both healthy and heart failure plasma samples. Important is the HNE Michael adduct, specifically identified in only heart failure samples. Moreover, the importance of stabilizing adducts is underlined by the fact that the acrolein Michael adduct could only be identified after reduction with NaBH4. Development of a cellular assay to determine pro-inflammatory activity of RAGE binders Another part of this project was focused on elucidating whether AGEs/ALEs induce an inflammatory response in cells. For this purpose, a collaboration was started with the Laboratory of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino. Using a rat epithelial lung cell line overexpressing RAGE, and a control cell line not expressing RAGE, it could be detected if AGEs/ALEs exhibit an effect by binding to RAGE. Moreover, both cell lines were transfected with a Nf-κB reporter gene allowing us a fast and sensitive method for determining if binding of RAGE induces a down-stream signaling pathway. This system implies a firefly luciferase gene downstream from the Nf-κB gene. When the Nf-κB pathway is activated, independently from RAGE, it produces the firefly luciferase enzyme. After adding a luciferin substrate, firefly luciferase is able to convert this substrate into another substrate with light as by-product, which can be measured by a luminometer. IL-1α was used as a positive control, since it induces a strong inflammatory response through Nf-κB. Moreover, known ligands of RAGE able to activate the Nf-κB pathway, were used to validate the cellular experiment, including HSA modified with fructose (AGE), and HMGB1. Results show that Nf-κB is already increased in untreated cells with RAGE and that AGEs induce the Nf-κB pathway independently from RAGE. Moreover, the difference between control and RAGE cells is not significantly increased in the presence of HMGB1 compared to untreated. However, the positive control seemed to induce a much stronger activity in cells with RAGE. Overall, this cellular assay is good for assessing pro-inflammatory activity, however, it is not optimized yet for distinguishing a RAGE-dependent mechanism. CONCLUSION In summary, by using an integrated MS (intact protein and bottom-up approach) and computational approach we have found that some ALEs generated from lipid peroxidation RCS are RAGE binders. We have also found the basic features that ALEs from HNE, MDA and ACR must have to be a RAGE binder: 1) the covalent adducts should greatly reduce or abolish the basicity of the target amino acid, 2) the basic amino acid should be at the center of a set of carboxylic acids which, once the residue is modified, become available to freely contact the RAGE positive residues. Next step was to use the VC1 technique to enrich AGEs/ALEs in biological samples. First, oxidized human plasma was used, however, using the Orbitrap LTQ XL, it was not sufficient to identify AGEs/ALEs. Therefore, analysis was moved to a higher resolution mass spectrometer, which allowed us to identify AGEs/ALEs in plasma samples of heart failure patients, showing the powerfulness of this new generation MS. Important was to understand whether ALEs could induce pro-inflammatory activity through RAGE, since we showed that ALEs are RAGE binders. Unfortunately, the cellular assay that was set up is efficiently in determining Nf-κB dependent pro-inflammatory activity, but not if it is RAGE dependent.

Le rôle important des facteurs de transcription tels que STAT3 et NF-kB dans les processus biologiques, ainsi que leur implication dans l’oncogenèse, justifient les nombreuses études consacrées à ces facteurs. Inhiber leurs activités semble présenter un grand intérêt thérapeutique. Dans un premier temps, nous avons montré que STAT3 est activé dans le cancer du côlon et qu’il est associé à des facteurs histopronostiques. Dans un deuxième temps, nous nous sommes intéressés à l’inhibition de facteurs de transcription STAT3 et NF-kB par des oligonucléotides leurres (ODN) comportant les séquences consensus de ces facteurs. Les ODN leurres induisent la mort d’une lignée cellulaire de cancer du côlon dont la croissance est sous la dépendance de STAT3 et NF-kB. Cependant, l’ODN anti-STAT3 peut interagir avec STAT1 et bloquer l’action de l’IFN. Ainsi, il est intéressant d’inhiber des facteurs de transcription dans les cellules tumorales mais les questions de spécificité ne sont pas résolues
The important role of transcription factors such as STAT3 and Nf-kB in biological processes, and their involvement in oncogenesis, justifies the numerous studies on these factors. To inhibit and control their activities appears to be promising therapeutic approach. Firstly, we have shown that STAT3 is constitutively activated in colon cancer and is associated with histopronostics features. In secondly, we inhibited the transcription factors STAT3 and NF-kB in cancer cell lines, we using decoy oligonucleotide containing the consensus target ssequences of these two factors. The decoy oligonucleotides induce the death of a cell line of colon cancer (SW480), however, the decoy ODN of STAT3 was found to interact with STAT1 and prevent IFNy action. Thus, it's of interest to inhibit transcription factors in tumor cells but specific issues are not resolved

碩士
國立嘉義大學
生物醫藥科學研究所
98
The recent research found that NF-kB, a transcription factor, can transcribe about 150 categories of proteins which regulate many cellular physiological responses, such as cellular inflammation, growth and apoptosis. Therefore, NF-kB is an important transcription factor. Previous studies indicated glutathionylation of protein can inhibit protein activities. Recently, our study found cinnamaldehyde can increase GSH in ECs, which inhibits TNF-a-induced NF-kB activity in ECs. Therefore, this research will investigate GSSG and H2O2/GSH in TNF-a-induced ECs and discuss protein glutathionylation on NF-kB-p65. We found GSSG、H2O2/GSH and NAC can increase total glutathionylated protein and NF-kB-p65 glutathionylation. Moreover, GSSG and H2O2/GSH inhibit TNF-a-induced ICAM-1 expression in transcriptional level by decrease NF-kB-p65 translocation. In addition, GSSG and H2O2/GSH inhibit NF-kB-p65 translocation independent with IkB-a degradation. 　　Taken together, these data suggest GSSG and H2O2/GSH decrease NF-kB-p65 translocation by increasing NF-kB-p65 glutathionylation. Therefore, We also transfected with GFP-p65 wild-type and GFP-p65 mutation c38s、c120s、c160s、c216s and then treat with GSSG to analyze NF-kB promoter activity. The result show NF-kB-p65 cystein 38、160、216 mutation will change GSSG inhibits TNF-a-induced NF-kB-p65 activation. To summarize, this research verifies that glutathionylation of NF-B-p65 in cystein 38、160、216 will affect the NF-kB-p65 protein activity.

碩士
國立嘉義大學
微生物與免疫學系研究所
97
During early atherogenesis, the endothelial cells excess produces adhesion molecules, which promote adhesion of monocytes to endothelial cells. It is causing formation of atheromatous plaque. Thus these adhesion molecules play an important role in the development of this disease. Previous studies have indicated that carnosol is a major ingredient of extracts from rosemary which exhibit anti-carcinogen and anti-inflammatory properties, but the underlying mechanisms are largely undefined. In this study, we examine the effects of carnosol and rosemary essential oil on TNF-α-induced adhesion molecules expression in endothelial cells. We found that rosemary essential oil and carnosol inhibit the expression of TNF-alpha-induced ICAM-1. Moreover, the carnosol was found to abrogate the adhesion of TNF-α-induced monocytes to endothelial cells. TNF-α-induced ICAM-1 expression is via NF-�羠 singling pathways. Our study showed that Carnosol inhibits TNF-α-induced p65 translocation by blocking the degradation of the IkappaB-�� in short-term pretreatments, but not in long-term pretreatments. Our findings showed that carnosol inhibits TNF-α-induced IKK phosphorylation in 3 hours pretreatments. Moreover, carnosol inhibits p65 translocation in 12 hours pretreatments, that blocked by Nrf2 siRNA, these results indicated that the long-term anti-inflammatory of carnosol was associated with the Nrf2-related genes. Carnosol induced the Nrf2-related gene expression, including heme-oxygenase (HO-1), over long-term pretreatments. Treating endothelial cells with HO-1 siRNA, blocks the anti-inflammatory effects of carnosol, these results showed that treatment of carnosol induces HO-1 expresssion is involved in the long-term anti-inflammatory. Moreover, we found treating of endothelial cells with carnosol significantly induces translocation of Nrf2 into the nucleus, increases expression of glutamate-cysteine ligase (GCL), and cellular glutathione (GSH) level in long-term pretreatments. Treating endothelial cells with buthionine sulfoximine (BSO), a specific inhibitor of glutamate-cysteine ligase, blocks the anti-inflammatory effects of carnosol and carnosol induced p65 glutathionylation. These results indicated that carnosol suppresses TNF-α-induced ICAM-1 expression through blocking the phosphorylation of IKK in short-term pretreatments, whereas it does so via the induction of HO-1 and GSH in long-term pretreatments.

碩士
高雄醫學大學
醫學研究所
100
Chronic inflammation is often evolved into the multiple malignancies. Many literatures mentioned that interleukin-6 (IL-6) and NF-κB participate in the inflammatory and cancer processes. Intestine-specific homeobox （ISX）is a newly identified homeobox transcription factor, its expression has been linked to liver tumorigenesis. In this study, we investigated whether NF-κB can respond to IL-6 and regulate ISX gene transcription. A luciferase reporter assay should that the -260/-230 is an IL-6 responsive region. Next, we performed a nucleus/cytosol fraction analysis to examine the shuttle of IKKα and NF-κB subunits（p65, p50, p52, Rel-B, c-Rel）. It showed that only p65 shuttle can respond to IL-6 treatment. In addition, it is known that the location of activated ISX is in nucleus. We further identified potent phosphorylated site on ISX that affects the location of ISX in nucleus by immunofluoresence assay.

博士
國立陽明大學
生物醫學影像暨放射科學系
101
Here a human hepatocellular carcinoma Huh7/NF-kB-tk-luc2/rfp cell clone was established using NF-kB responsive element as a promoter to drive herpes simplex virus thymidine kinase (tk) and firefly luciferase (luc2) dual reporter genes for monitoring NF-kB activity, and co-transfected with red fluorescent protein gene (rfp) for identifying the viable tumor cells. With this Huh7/NF-kB-tk-luc2/rfp tumor-bearing animal model combined with multimodalities of molecular imaging, simultaneous imaging of temporal changes of NF-kB activity and viable tumor cells during sorafenib treatments could be performed. Sorafenib is a multikinase inhibitor approved by FDA for the treatment of renal carcinoma via Ras-Raf-MAPK-MEK-ERK pathway, and was found to be beneficial for other types of cancers as well, especially hepatoma. NF-kB responsive element with tk-luc2 dual reporter genes were used to combined with multimodalities of molecular imaging, such as bioluminescent imaging (BLI) for tracking the real-time NF-kB activity, and rfp gene for the imaging (RFPI) assay of viable tumor cells during sorafenib treatment. The NF-kB activity has been reported to be associated with chemo- and radioresistance during cancer treatments. The results showed that sorafenib could suppress the NF-κB/DNA binding activity, and the expression of downstream effector proteins to enhance tumor cell killing through inhibition of anti-apoptotic pathway, tumor cell proliferation, and invasion, while increase apoptosis of tumor cells. In addition, the relative photon fluxes obtained from RFPI and BLI, each represented the viable tumor cells and cells with NF-κB activation, respectively, decreased after sorafenib treatment by 50 to 65%, and 87.5 to >90%, suggesting that the percentage of suppression of NF-κB activation was much higher than that of HCC tumor cell killing in the tumor mass at anytime during sorafenib treatments. In conclusion, simultaneous molecular imaging of the temporal change of NF-κB activity and the viable tumor cells in the same Huh7/NF-κB-tk-luc2/rfp tumors of the animal may reflect the real status of NF-κB activity and the viable tumor cells at the time of imaging. This system may be applied for screening the potential chemotherapeutic drugs, and for the therapeutic efficacy evaluation of new preclinical trials, which using NF-κB as a major target.

碩士
國立臺灣大學
生化科學研究所
88
Pregnancy-specific glycoproteins (PSGs) are primarily expressed in the placenta and become the major glycoproteins at term. To understand the function and regulation of PSG regulation in pregnancy, the promoter elements and cDNA sequences of a rodent PSG gene have been characterized. The cDNA of the rat pregnancy gene, rnCGM3, is 2761bp in length and contains an open reading frame that encodes a 475 amino acid polypeptide. The transcription initiation site of rnCGM3 is located at nucleotide —197 upstream of the translation start site. Three nuclear protein binding sites: FPⅠ, FPⅡ and FPⅢ in 5’-flanking region of rnCGM3 gene have been characterized. The FPⅡ binding factor was shown to be C/EBPβ. RBPJκ was found to be the factor binds to both FPⅠ and FPⅢ. We also found another important transcription factor, the p65 subunit of NF-κB, which binds to the FPⅢ element in the yeast one-hybrid system. In this study, we showed that both p50 and p65 subunits of NF-κB bind to the FPⅢ element in the electrophoretic mobility shift assay (EMSA). A core sequence, 5'-GGGAAA-3', on the FPⅢ element for NF-κB was defined by mutagenesis analysis. Based on EMSA with NF-κB-enriched nuclear extracts from 293 cells transfected with different subunits of NF-κB, we found that both mono- and heterodimer of NF-κB bind to FPⅢ. We further studied the effect of two different transcription factors, NF-κB and RBPJκ, on the regulation of rnCGM3 gene expression. By transient expression analyses, we found the expression of CAT reporter gene could be activated by p65 and this stimulatory effect of p65 could be repressed by RBPJκ. Our results suggest that rnCGM3 gene expression may be mediated by the NF-κB and the Notch signaling pathway via RBPJκ.

博士
長庚大學
臨床醫學研究所
89
Tumor necrosis factor (TNF) is a multifunction cytokine that plays important roles in the program cell death, inflammatory response and viral clearance. On the othre hand, TNF may promote cell regeneration by activate Nuclear factor kappB (NF-kB) transactivation pathway. The cytotoxicity induced by TNF plays an important role in the pathogenesis of chronic hepatitis B and C. The activation of NF-kB may inhibit apoptosis that may faccilitate viral persistent and hepatocarcinogenesis. For better understanding the differences of persistent infection and pathogenesis between chronic hepatitis B and C, we studied 38 patients with chronic hepatitis B, 40 with chronic hepatitis C, 9 with dual hepatitis B and C for cytokine levels and compared with 12 healthy controls. Plasma levels of TNFa, soluble TNF receptor-I (TNFR-I), soluble TNF receptor-II (TNFR-II) and related cytokines were quantified by ELISA and correlated with histology activity index. Expressions of TNFR-I and II in liver tissues were also examined in 33 with HBV and 15 with HCV by semi-quantitative reverse transcription- polymerase chain reaction (RT-PCR). The results showed that sTNFR-I levels correlated with liver inflammation in the whole study patients. Type 1 helper T cells (Th1) cytokines levels were higher in the groups with higher inflammatory activities. Most importantly, sTNFR-I levels correlated with liver inflammation in HBV, whereas sTNFR-II levels correlated with liver inflammation in HCV. The expressions of TNFR-I and -II messanger RNA (mRNA) in liver tissues were similar and comparable between hepatitis B virus (HBV) and hepatitis C virus (HCV). These findings suggest that TNFR signal transduction pathway is differentially modulated between HBV and HCV infection, and may be relevant to immunopathogenesis. The HCV core protein is also a multifunctional protein. It may bind to the death domain of TNFR-I, and to the cytoplasmic tail of lymphotoxin-b receptor, implying that it may be involved in the apoptosis and anti-apoptosis signaling pathways. To address this issue, electrophoretic mobility shift assay and immunohistochemical studies were used to demonstrate the activation of NF-kB in HCV-infected liver tissues and in HCV core-transfected cells. The activation of NF-kB was correlated with the apoptosis assays. The results showed that NF-kB activation could be demonstrated in HCV-infected livers and HCV core-transfected cells. The data of electromobility shift assay (EMSA) correlated with those of immunohistochemical studies which revealed a higher frequency of NF-kB nuclear staining in HCV-infected than in normal livers. NF-kB activation conferred resistance to TNFa-induced apoptosis in HCV core-transfected cells. Inhibition of NF-kB activation by pyrrolidine dithiocarbamate sensitized them to TNFa-induced apoptosis. These findings suggest that HCV infection may cause anti-apoptosis by activation of NF-kB and implicate a mechanism by which HCV may evade the host's immune surveillance leading to viral persistence and possibly to hepatocarcinogenesis. The NF-kB activation status in hepatocellular carcinoma (HCC) has not yet been explored, therefore. immunohistochemical stainings were done on formalin fixed liver tissues from 65 HCCs and 9 normal control subjects to search for active nuclear RelA and nuclear IkBa proteins. Nuclear extract from 37 pairs of fresh frozen tumor and non-tumor liver tissues and 7 normal controls were tested for NF-kB-DNA binding activity by electrophoretic motility shift assay. The RelA and IkBa protein expressions were studied by Western blotting. Nuclear NF-kB stainings were significantly more abundant in HBV or HCV infected tumor as well as non-tumor part of HCC than in normal controls. Nuclear NF-kB-DNA binding activity and nuclear RelA proteins expression were higher in tumor than in non-tumor tissues; whereas, cytosolic IkBa protein expression was generally higher in non-tumor than in tumor tissues. Constitutive activation of NF-kB was found more frequently in tumor than in non-tumor tissues. NF-kB overexpression accompanied by dysregulation of IkBa may play a role in hepatocarcinogenesis of HBV or HCV infection.

博士
國防醫學院
生命科學研究所
86
Epstein-Barr virus (EBV) is associated with many malignant tumors including nasopharyngeal carcinoma (NPC). Previous stuides have indicated that EBV-encoded oncoprotein, latent membrane protein 1 (LMP 1), is expressed in many NPC tissues. LMP 1 has been shown to stimulate HIV LTR through the two NF-kB binding sites within this promoter. In this thesis, I tested the feasibility of using this property of LMP 1 as a therapeutic strategy for treatment of NPC. In appendix of this thesis, I took advantage of the retroviral trapping sysytem to screen for new cellular factors that could be activated or inhibited by LMP 1 of EBV. The therapeutic strategy combines preferential killing of the LMP 1-expressing cells by gene transfer of NF-kB-mediated herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir(GCV) treatment. The 800-bp HIV-LTR, which contains two NF-kB binding sites, was used to drive the HSV-tk gene. C33A and NPC076 cell clones stably expressing the LMP 1 and the HSV-tk genes were subjected to the GCV sensitivity test. Results showed that cells expressing both the LMP 1 and the HSV-tk genes were highly sensitive to GCV treatment. These cells were introduced into nude mice subcutaneously and tumors became plapable within 2 weeks. GCV was then introduced intraperitoneally to these mice and the sizes of the tumors were measured daily.Results showed that the tumors regressed in the group of mice carrying cells that stably expressed both the LMP 1 and the HSV-tk genes, but not in mice carrying cells containing LMP 1 or HSV-TK alone. Date indicate that the HSVtk gene expressed from a NF-kB-binding motif-containing promter that is regulated by LMP 1 may be used as an in vivo gene therapeutic strategy for EBV LMP 1-expressing cancers such as NPC.

No
A novel function of NF-KB in the development of most ectodermal appendages, including two types of murine pelage hair follicles, was detected in a mouse model with suppressed NF-KB activity (CI¿B¿¿N). However, the developmental processes regulated by NF-¿B in hair follicles has remained unknown. Furthermore, the similarity between the phenotypes of CI¿BA¿N mice and mice deficient in Eda A1 (tabby) or its receptor EdaR (downless) raised the issue of whether in vivo NF-KB regulates or is regulated by these novel TNF family members. We now demonstrate that epidermal NF-KB activity is first observed in placodes of primary guard hair follicles at day E14.5, and that in vivo NF-KB signalling is activated downstream of Eda A1 and EdaR. Importantly, ectopic signals which activate NF-KB can also stimulate guard hair placode formation, suggesting a crucial role for NF-KB in placode development. In downless and CI¿B¿¿N mice, placodes start to develop, but rapidly abort in the absence of EdaR/NF-KB signalling. We show that NF-KB activation is essential for induction of Shh and cyclin D1 expression and subsequent placode down growth. However, cyclin D1 induction appears to be indirectly regulated by NF-KB, probably via Shh and Wnt. The strongly decreased number of hair follicles observed in CI¿B¿¿N mice compared with tabby mice, indicates that additional signals, such as TROY, must regulate NF-KB activity in specific hair follicle subtypes.