Дисертації з теми "NF-kappa B pathway"

Thesis (Ph.D.)--Cleveland State University, 2008.<br>Abstract. Title from PDF t.p. (viewed on Oct. 6, 2008). Includes bibliographical references (p. 213-240). Available online via the OhioLINK ETD Center. Also available in print.

Cancer is one of the major causes of morbidity in the world. Although the overall survival of cancer has been significantly improved by chemotherapy in the last three decades, the success of cancer chemotherapy is still severely limited by the lack of selectivity of anti-cancer drugs to malignant cells leading to dose-limiting toxicity and the resistance of cancer cells to the conventional anti-cancer drugs. Gene-directed enzyme prodrug therapy (GDEPT) was designed to direct the anti-cancer drugs to specifically target the cancer cells by using cancer specific promoter to drive the expression of enzyme which can convert prodrug into anti-cancer drug specifically in cancer cells. However, this strategy is hindered by the lack of strong cancer specific promoters to specifically express drug-converting enzymes in cancer cells. In consequence, there is not enough anti-cancer drug activated inside the cancer cells. The first part of this study was to employ NF-κB binding sites as a novel enhancer system to improve the promoter activity of carcinoembryonic antigen (CEA) and human telomerase reverse transcriptase (hTERT) for GDEPT. In this system, the basal CEA promoter sequences were placed downstream of the 4 or 8 NF-κB DNA binding sites linked in tandem (κB4 or κB8). The system was designed to serve two particular purposes: to exploit the high levels of intratumoural NF-kB expression and keep the relative tumour specificity of the CEA and hTERT promoters. The results demonstrated that κB enhancer systems increased the transcriptional activity of CEA and hTERT promoter without compromising its cancer specificity. The fidelity of the κB4-CEA enhancer-promoter system was therefore improved by the increased transcriptional contrast between the cancer and normal cells. Moreover, in comparison with CEA promoter alone, κB-CEA enhancer-promoter system expressed human thymidine phosphorylase (TP) protein at significantly higher levels which were comparable to those expressed by CMV promoter. The κBCEA- TP system transfected cells demonstrated significantly higher sensitivity to 5'-Deoxy-5-Fluorouridine (5'-DFUR), a prodrug of 5-fluorouracil (5-FU). The second part of this study was involved in using NF-κB inhibitor as a chemosensitizer to sentizise the anti-cancer drug-induced chemoresistance cells to anti-cancer drugs. The results derived from this study manifested that the anti-alcoholism drug, Disulfiram (DS), and anti-inflammatory drug, triptolide (PG490), markedly enhanced the cytotoxicity of several conventional anti-cancer drugs in colon, lung and breast cancer cell lines. PG490 induced caspase-dependent cell death accompanied by a significant decrease in Bcl-2 levels. PG490 induced the expression of p53 and down-regulated p21 expression. This study indicated that some clinically used non-cancerchemotherapeutic drugs may be developed as chemosensitizers for cancer chemotherapy

On a montré ici que les voies canonique/non-canonique de NF-B ne sont pas indépendantes et qu’un rôle principal de la voie canonique est l’activation des acteurs de la voie non-canonique. La traduction est requise et la stimulation par LPS régule positivement un éventuel intermédiaire dans la signalisation par LPS/CD40 qui supporte l'activation de la voie non-canonique. De plus, la voie non-canonique est constitutivement activée dans les cellules de HL, impliquant la phosphorylation persistante de p100. L'activation transitoire et constitutive de la voie non-canonique implique l'incorporation de p100 et p52 dans un complexe « megadalton ». TAP a permis d’identifier des partenaires interagissant avec p100. EDD (une molécule identifiée) induit le processing de p100 par co-expression. La formation du complexe de TRAF3 (autre facteur identifié) avec p100 est médiée par NIK et requiert son activité kinase. L'expression de NIK favorise le recrutement de TRAF3/p100 au signalosome p100<br>It is shown here that the canonical/non-canonical NF-B pathways are not independent and a master regulatory role of the canonical pathway is to upregulate activators of the non-canonical pathway. The data further implicate a translation requirement and that LPS stimulation upregulates a potential intermediate in LPS/CD40 signaling which supports activation of the non-canonical pathway. Further, the non-canonical pathway is constitutively activated in HL cells, involving persistent p100 phosphorylation. It was found that transient and constitutive activation of the non-canonical pathway involves incorporation of p100 and p52 into a megadalton complex. TAP was employed to identify novel p100 interacting partners. EDD (an identified molecule) induces processing of p100 upon co-expression. The complex formation of TRAF3 (another p100 interactor) with p100 is mediated by NIK and requires its kinase activity. The expression of NIK promotes recruitment of TRAF3/p100 to the p100 signalosome

Metastatic melanoma is an aggressive, incurable cancer with historically few therapeutic options. The discovery that 60% of melanomas harbor the oncogenic BRAF_V600E mutation, which constitutively activates the MAPK pathway, has provided a promising new therapeutic axis. Although MAPK pathway inhibitor therapy has shown striking clinical results in BRAF_V600-mutant melanoma, this approach faces three limitations. First, 10-20% of BRAF_V600-mutant melanomas never achieve meaningful response to MAPK pathway inhibitor therapy (intrinsic resistance). Second, among BRAF_V600-mutant melanomas initially responding to MAPK pathway inhibitor therapy, relapse is universal (acquired resistance). Third, approximately 40% of melanomas lack BRAF_V600 mutations and so are not currently candidates for MAPK pathway inhibitor therapy. We sought to address each of these problems: by characterizing the phenomenon of intrinsic MAPK pathway inhibitor resistance, by finding ways to perturb mechanisms of acquired MAPK pathway inhibitor resistance, and by identifying novel dependencies in melanoma outside of the MAPK pathway. Intriguingly, the NF-kappa B pathway emerged as a common theme across these investigations. In particular, we establish that MAPK pathway inhibitor sensitive and resistant melanomas display distinct transcriptional signatures. Unlike most BRAF_V600-mutant melanomas, which highly express the melanocytic lineage transcription factor MITF, MAPK pathway inhibitor resistant lines display low MITF expression but high levels of NF-kappa B signaling. These divergent transcriptional states, which arise in melanocytes from aberrant MAPK pathway activation by BRAF_V600E, remain plastic and mutually antagonistic in established melanomas. Together, these results characterize a dichotomy between MITF and NF-kappa B cellular states as a determinant of intrinsic sensitivity versus resistance to MAPK pathway inhibitors in BRAF_V600-mutant melanoma. In separate investigations, we have shown that, NFKB1 p105, a member of the NF-kappa B family, intimately regulates levels of COT, a known effector of resistance to MAPK pathway inhibitors. Moreover, we have used shRNA screening to nominate particular nodes within the NF-kappa B pathway, including MYD88 and IRF3, as candidate melanoma lineage-specific dependencies. Cumulatively, although these studies use diverse approaches to investigate the limitations of MAPK pathway inhibitor therapy in melanoma, they converge in nominating the NF-kappa B pathway as a previously underappreciated feature of melanoma biology and suggest the relevance of this pathway for future investigation.

Ovarian cancer is one of the most deadly female malignancies. Despite advances in the treatment of ovarian cancer for the past decade, the cure rate of this disease is moderately improved. Emerging evidence suggests the molecular personalized therapeutic approach become popular for human cancer treatment. The nuclear factor-kappa B (NF-κB) signaling pathway has been shown to play multiple roles in cancer progression such as anti-apoptosis, cell cycle, angiogenesis and metastasis. This study attempted to characterize the functional roles of transforming growth factor (TGF)-β-activating kinase 1 (TAK1) in the activation of NF-κB signaling. Importantly, this study provided evidence showing the significance of TAK1-NF-κB signaling axis in ovarian cancer aggressiveness during omental metastasis. Using quantitative RT-PCR and immunohistochemical analyses, TAK1 was frequently up-regulated and was significantly associated with high-grade (P=0.001), lymph node and distant metastasis (P=0.025), as well as a tendency toward advanced stage ovarian cancers (P=0.08). Functionally, enforced expression of TAK1 could augment cell proliferation, colony formation, anchorage-independent growth ability and migration/invasion in ovarian cancer cells. Conversely, repression of TAK1 expression by genetically or pharmaceutical approach abrogated these tumorigenic capacities including tumor growth in vivo. Furthermore, co-treatment of (5Z) -7-Oxozeaenol could sensitize ovarian cancer cells to cisplatin-induced cell apoptosis, indicating TAK1 is also involved in chemoresistance. Mechanistically, using Western blotting and NF-κB -reporter luciferase analyses, the elevation of TAK1 phosphorylation at Ser412 but not Thr184/187 was found to associate with the activation of NF-κB in ovarian cancer cells solely. A series of functional studies with genetic and pharmaceutical alterations revealed that the increased TAK1 Ser412 phosphorylation was required for exerting the ovarian cancer cell oncogenesis. Omental metastasis is the common phenomenon observed in most of advanced-stage ovarian cancer. Using omentum conditioned medium (OCM), the findings of this study showed that the omentum tissue was able to secrete numerous factors including chemokines such as GRO-α and IL8 in activating TAK1-NF-κB signaling cascade, which thereby induced increased oncogenic capacities in cell growth, migration and invasion. Taken together, this study suggests that TAK1-NF-κB signaling axis is indispensable for promoting oncogenesis of ovarian cancer and targeting this pathway may be a promising personalized cancer therapeutic approach in ovarian cancer.<br>published_or_final_version<br>Obstetrics and Gynaecology<br>Doctoral<br>Doctor of Philosophy

This project contributed to a greater understanding of chemotherapy resistance in non-small cell lung cancer (NSCLC), a prevalent issue to chemotherapy. The potential of the inflammatory transcription factor NF-&#312;B as a therapeutic target was investigated to combat NSCLC chemo-resistance using in vitro and in vivo approaches. The activity of NF-&#312;B was elucidated in models of chemo-resistant NSCLC and where specific inhibitors of NF-&#312;B were used to block activity. In addition, a novel 3D model of chemo-resistance in NSCLC was generated which will serve as an important model in future studies.

2006/2007<br>Atherosclerosis, a progressive cardiovascular disease, is characterized by the accumulation of cholesterol in macrophage deposits (foam cells) and the formation of atherosclerotic plaques in the walls large- and medium- sized arteries. The earliest events in the development of atherosclerosis involve progressive modifications in the endothelial micro-environment. This endothelial dysfunction is a complex of multi-step mechanisms, for which reduced NO levels have been reported as a marker, is characterized by increasing expression of adhesion molecules (AMs), which mediate the diapedesis (migration) of inflammatory and immunocompetent cells through the endothelial layer into the arterial wall. NO is synthesized intracellularly by nitric oxide enzymes (eNOS and iNOS) and is regulated by a variety of stimuli. NO acts as an autocrine or paracrine hormone, as well as intracellular messenger, with a critical role in vascular endothelial growth factor-induced angiogenesis and vascular hyperpermeability in vitro. The over-expression of AMs is orchestrated by pro-inflammatory cytokines, particularly TNF-alpha. The two major subsets of AMs participating in these processes are the selectins, in particular E-selectin, and the immunoglobulin gene superfamily, in particular vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Transcriptional regulation of these inflammatory genes requires the participation of several proteins, inducible activators, as: NF-kappa B and (CRE)-binding protein (CREB). The most abundant form of NF-kappa B is an heterodimer of p50 and p65; which is sequestered in the cytoplasm in an inactive form through interaction with the I kappa B inhibitor proteins. Signals that induce NF-kappa B release dimmers to enter to the nucleus and induce gene expression. Pyrridoline dithiocarbamate (PDTC) a metal-chelating compound inhibits NF-kappa B by blocking ubiquitine ligase activity towards phosphorylated I kappa B, in turn downregulating the expression of E-selectin, VCAM-1 and ICAM-1. CREB is a widely expressed DNA-binding protein, downstream target of cAMP, activated by phosphorylation on serine 133. A regulatory site, on the gene promoters of both E-selectin and VCAM-1, binds both NF-kappa B and CREB transcription factors. Unconjugated bilirubin (UCB), long considered to be simply a waste end product of heme metabolism and a marker for hepatobiliary disorders, is now thought to function as an endogenous tissue protector by attenuating free radical-mediated damage to both lipids and proteins. There is increasing epidemiological evidence supporting an inverse association between cardiovascular disease and plasma levels of bilirubin. Recent studies indicated that bilirubin may be protective in the development of atherosclerotic diseases by inhibiting the proliferation of vascular smooth muscle cells by mechanisms yet to be established. It has been proposed that UCB can interfere with the atherosclerotic disease development by inhibiting the trans-endothelial vascular cell adhesion molecule (VCAM-1)-dependent migration of monocytes into the intima. The aim of this study is to investigate the effect of the UCB in the endothelial dysfunction. Specifically UCB effects on NO production, AMs expression and the regulatory transcription factors involve in the inflammatory response. Variable doses of free bilirubin (Bf) (the active form of UCB in plasma), simulating upper normal (15 nM) and modestly elevated levels (30 nM) for plasma, were evaluated in two models of endothelial cells. A) H5V, murine microvascular endothelial cell line, and B) HUVEC (Human Umbilical Vein Endothelial Cells), isolated from the vein of human umbilical cord. TNF-alpha (20 ng/mL) was added in order to reproduce, in vitro, the endothelial dysfunction and describe UCB contribution on its effects. UCB alone reduced the viability in H5V cells by MTT assay in a dose dependent manner after 24 hours while no effect was observed in the LDH released. In the first set of experiments NO production in H5V cells was evaluated, a time-depended increase on NO basal and a dose-dependent decrease on NO concentration after TNF-alpha (20 ng/mL) were observed. NO reduction related TNF-alpha was seen at all times studied. The effect of UCB was studied in co-treatments with TNF-alpha for 24 and 48 hours. UCB (Bf 15 and 30 nM) significantly reversed the reduction of nitrite content induced by TNF-alpha at 48 hours. The gene expression analysis was performed by Real Time PCR technology with specific primers for eNOS, iNOS, E-selectin, VCAM-1 and ICAM-1. In H5V cells, TNF-alpha increased the expression of all the genes studied (except eNOS) at 2, 6 and 24 hours. The co-treatment with UCB, at a Bf that did not themselves affect the expression of the three adhesion molecules, blunts the over-expression of E-selectin, Vcam-1 and iNOS induced by a pro-inflammatory cytokine such as TNF-alpha. The inhibitory effect of UCB was usually modest (20-30%) and detected at 2 and/or 6 hours, but had disappeared 24 hours. Furthermore, a synergistic effect between TNF-alpha and UCB was seen on the expression of iNOS at 24 hours, indicating a biphasic regulation. Moreover, no effects were seen on eNOS. Similar results were observed in the regulation of the gene expression of the AMs and viability in HUVEC cells, indicating the lack of species specific effect. However, no effect of TNF-alpha or UCB was seen in the expression of iNOS, eNOS or NO content. Western blot analysis in H5V cells confirmed that TNF-alpha induced the expression of E-selectin, VCAM-1 and ICAM-1 in a time-dependent manner. This effect was blunted after 24 hours by the presence of UCB (Bf 15 and 30 nM). The contribution of NF-kappa B pathway in UCB effects was investigated by addition of a specific inhibitor, PDTC. The co-treatment with PDTC and UCB for 2 hours produced an additive reduction of TNF-alpha effect on Eselectin, VCAM-1, and iNOS in H5V cells. In addition, UCB prevented the nuclear translocation of NF-kappa B induced by TNF-alpha. Failure of UCB to alter TNF-alpha -induced phosphorylation of CREB (at Ser 133) suggested that the CREB pathway was not involved in the UCB inhibition. The results obtained in the present study shows that unconjugated bilirubin, even at upper-normal physiological (15 nM) and mildly elevated (30 nM) Bf can modulate gene expression and endothelial cell function. Furthermore, UCB may regulate NO levels by a biphasic regulation of iNOS, and in addition influences the expression of the endothelial adhesion molecules. In summary, these data indicates that bilirubin limits the over-expression of the adhesion molecules and regulates the NO metabolism in the pro-inflammatory state induced by the cytokine TNF-alpha. Even though UCB alone does not alter these markers. UCB effects are mediated in part by a modulation of the NF-kappa B transcription factor. These results support the concept that modestly elevated concentrations of bilirubin may help prevent atherosclerotic disease as suggested by epidemiological studies.<br>La malattia cardiovascolare è la causa di morte e disabilità più importante nel mondo occidentale e tra le malattie cardiovascolari, quella vascolare aterosclerotica rappresenta la percentuale maggiore. Le lesioni aterosclerotiche si creano principalmente nelle arterie di medio e grande calibro con parete muscolare elastica e possono determinare ischemia nei tessuti a valle della lesione stessa. Numerose osservazioni epidemiologiche hanno riscontrato una significativa correlazione inversa tra incidenza di malattia cardiovascolare ischemica e livelli serici di bilirubina. Sono stati pubblicati studi di prevalenza su campioni di popolazione maschile e sulla popolazione generale, studi prospettici su soggetti con iperbilirubinemia non coniugata (Sindrome di Gilbert) o nella popolazione generale. Questi studi hanno confermato l'importanza dei valori di bilirubinemia nella valutazione del rischio d'incidenza di malattia ischemica coronarica. In alcuni casi, il valore di bilirubinemia è addirittura identificabile come fattore di rischio indipendente di malattia. Analoghe osservazioni sono state documentate nella prevalenza della malattia vascolare periferica aterosclerotica. L'endotelio è un tessuto in grado di rispondere ai vari stimoli con la produzione di sostanze vasoattive, tra cui l'ossido nitrico (NO) sintetizato principalmente dagli enzimi ossido nitrico sintetasi (iNOS ed eNOS), e le molecole di adesione (AMs), E-selectina, VCAM-1 e ICAM-1. Il primo evento che si verifica nella insorgenza di una lesione aterosclerotica è la comparsa della disfunzione endoteliale. Per disfunzione endoteliale si intende una successione di condizioni patologiche che coinvolgono l'endotelio vascolare determinate da una ridotta biodisponibilità di NO e un aumento dell'espressione delle molecole di adesione. Questi eventi favorirebbero l'adesione di linfo/monociti e la successiva migrazione nell'intima. Una riduzione di NO e l'incremento dell'espressione dalle AMs nelle cellule endoteliali stesse avviene nel processo infiammatorio attraverso l'induzione di fattori di trascrizioni tra cui NF-kappa B e CREB. Questo progetto di ricerca si pone come obiettivo lo studio degli effetti della bilirubina non coniugata (UCB) a concentrazioni fisiologiche normali o lievemente incrementate sull'insorgenza della disfunzione endoteliale, prima manifestazione della malattia aterosclerotica. Specificamente, studiare l'effetto della UCB libera (Bf), che è la porzione attiva di UCB in plasma non legata all'albumine, nella regolazione della produzione di NO e l'espressione delle AMs. Lo studio è stato condotto utilizzando due modelli in vitro di cellule endoteliali. a) H5V, linea cellulare immortalizzata di topo e b) HUVEC cultura primaria di cellule umane, della vena ombelicale (Human Umbilical Vein Endothelial Cells). La citochina TNF-alpha (20 ng/mL) è stata utilizzata per indurre lo stato di disfunzione endoteliale. Lo studio ha evidenziato che livelli elevati di bilirubina non coniugata libera (Bf 100 nM) riescono a ridurre significativamente la vitalità cellulare valutata attraverso il test MTT senza provocare variazioni nella liberazione di LDH. Inoltre è stato evidenziato un incremento basale del livello extracellulare di NO in maniera tempo dipendente. Il trattamento con TNF-alpha provoca una riduzione dose dipendente del livello extracellulare di NO a tutti i tempi di trattamento. UCB riesce a modulare l'effetto di TNF-alpha dopo 48 ore di cotrattamento. Questo effetto si correla con un incremento nell'espressione genica di iNOS dopo 24 ore. A per tempi brevi di trattamento (2 e 6 ore) UCB riduce l'espressione genica di E-selectina, VCAM-1 in entrambi tipi cellulari, dimostrando l'assenza di un effetto specie-specifico. Inoltre è stata anche osservata, nelle cellule H5V una riduzione dell'espressione di iNOS dopo 2 ore. Alla riduzione dell'espressione genica delle AMs segue la riduzione della espressione proteica dopo 24 ore di trattamento nelle cellule H5V. Un effetto inibitorio additivo all'azione dell'UCB si è ottenuto inibendo in modo specifico il fattore di trascrizione NF-kappa B con PDTC. In seguito a trattamento con UCB si è riscontrata una riduzione della localizzazione di NF-kappa B nel nucleo e nessun effetto sulla fosforilazione del fattore di trascrizione CREB. Questi risultati confermano il coinvolgimento di UCB a concentrazioni fisiologiche normali (Bf 15 nM) o lievemente elevate (Bf 30 nM) nella regolazione dal metabolismo del NO e nella modulazione dell'espressione delle AMs indotte da TNF-alpha, anche se UCB non influisce di per sé su codesti marcatori. Questo lavoro conferma che l'effetto della bilirubina, in moderate concentrazione fisiologiche, è coinvolta nella prevenzione delle malattie aterosclerotiche, secondo quanto suggerito dagli studi epidemiologici.<br>1977

The cytokine tumor necrosis factor α (TNFα) stimulates the NF-кB, SAPK/JNK, and p38 mitogen-activated protein (MAP) kinase pathways by recruiting Rip1 and Traf2 proteins to the tumor necrosis factor receptor 1 (TNFR1). Genetic studies have revealed that Rip1 links the TNFR1 to the IкB kinase (IKK) complex, whereas Traf2 couples the TNFR1 to the SAPK/JNK cascade. We found TNFα-induced p38 MAP kinase activation and interleukin-6 (IL-6) production is impaired in rip1-/- murine embryonic fibroblasts (MEF) but unaffected in traj2-/- MEF, demonstrating that Rip1 is also a specific mediator of the p38 MAP kinase response to TNFα. Moreover, we demonstrate that endogenous Rip1 associates with the MAP3K, Mekk3 in response to TNFα and that TNFα-induced p38 MAP kinase activation is impaired in mekk3-/- cells, indicating that Rip1 may mediate the p38 MAP kinase response to TNFα by recruiting Mekk3. We also demonstrate that Rip1 is phosphorylated and ubiquitinated in response to Tnfα and that Rip1 phosphorylation is not required for ubiquitination of Rip1. Furthermore, TNFα-induced ubiquitination of Rip1 is impaired in Traf2-/- cells, suggesting that Traf2 is the E3 ubiquitin ligase responsible for the TNFα-dependent ubiquitination of Rip1. Finally, recruitment of the ubiquitinated Tak1 complex is dependent on the presence of Rip1, suggesting that Rip1 ubiquitination rather than its phosphorylation is critical in TNFR1 signaling.

Thesis (Ph. D.)--University of California, Davis, 2005.<br>Degree granted in Comparative Pathology. On t.p. "[kappa]" appears as Greek letter. Also available via the World Wide Web. (Restricted to UC campuses)

Intestinal epithelial differentiation entails the formation of highly specialized cells with specific absorptive, secretory, digestive and immune functions. Cell-cell and cell-microenvironment interactions appear to be crucial in determining the outcome of the differentiation process. Using the Caco-2 cell line that can undergo spontaneous differentiation when grown past confluency, we observed a loss of VCAM1 (vascular cell adhesion molecule-1) expression while ICAM1 (intercellular cell adhesion molecule-1) expression was seen to be stable in the course of differentiation. Protein kinase C theta (PKC&theta<br>) acted downstream of PKC to inactivate Inhibitor of kappa B (IB) and activate NF-&kappa<br>B in the undifferentiated cells and this axis was inhibited in the differentiated cells. The increase in ICAM1 expression in the differentiated cells was due to a transcriptional upregulation by C/EBP. The protein expressions of both ICAM-1 and VCAM-1, however, were found to decrease in the course of differentiation, with both proteins getting post-translationally degraded in the lysosome. Functionally, a decrease in adhesion to HUVEC cells was observed in the differentiated Caco-2 cells. Thus, the regulation of ICAM-1 and VCAM-1, although both NF-B target genes, appear to be different in the course of epithelial differentiation. microRNAs are known to regulate many cellular pathways. miR-146a, which is known to target NF-&kappa<br>B, was shown to be highly upregulated in differentiated Caco-2 cells. As a predicted target of miR-146a, mRNA and protein expression of MMP16 was inversely correlated with miR-146a during differentiation of Caco-2 cells. miR-146a could bind to the 3&rsquo<br>UTR of MMP16 and ectopic expression of miR-146a resulted in a decreased mRNA and protein expression of MMP16 in the undifferentiated Caco-2 and HT-29 cells. Functionally, decreased gelatinase activity determined by gelatin zymography and reduced invasion and migration through Transwells was observed. In the final part of the thesis, the inhibition of NF-&kappa<br>B via PPAR&gamma<br>in 15-Lipoxygenase-1 (15LOX1) expressing cells was investigated. The expression of 15LOX1, a member of the inflammatory arachidonate cascade, could lower phosphorylation of I&kappa<br>B&alpha<br>and NF-&kappa<br>B DNA binding activity which was reversed with a 15LOX1 inhibitor. This inhibition was mediated by phospho-PPAR&gamma<br>, which in turn was phosphorylated by ERK1/2.

Dissertation (Ph.D.)--University of Toledo, 2007.<br>"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 72-96.

Indiana University-Purdue University Indianapolis (IUPUI)<br>S-nitrosoglutathione reductase (GSNOR) also known as glutathione- dependent formaldehyde dehydrogenase (FDH), is a zinc-dependent dehydrogenase. GSNOR oxidizes long chain alcohols to an aldehyde with the help of a molecule of NAD+. GSNOR was initially identified as FDH because of its role in the formaldehyde detoxification pathway. The only S-nitrosothiol (SNO) substrate recognized by GSNOR is GSNO. A transnitrosation reaction transfers NO from nitrosylated proteins or S-nitrosothiols (RSNO) to glutathione to form S-nitrosoglutathione. This GSNO is finally converted to glutathione disulfide (GSSG) by a two step mechanism. Cellular GSNO is a nitric oxide reservoir that can either transfer to or remove from the proteins a NO group. Reduction of GSNO by GSNOR depletes this reservoir and therefore indirectly regulates protein nitrosylation. GSNOR inhibitors which can increase the basal GSNO levels will be another potential therapy. Several GSNOR inhibitors were identified in our laboratory and the aim of this study was to understand their cellular effects. One of the experiments studied the effect of the compound on protein-SNO. The role of nitric oxide in regulation of NF-κB pathway is reviewed by Bove and van der Vliet. We focused on identification of nitrosylated proteins using protein specific antibodies. We identified nitrosylation of IKKβ. So the question raised was whether nitrosylation of IKKβ affects its activity. IKKβ is responsible for phosphorylation of IκBα and phosphorylation of IκBα results in its degradation and activation of NF-κB pathway. Therefore, we studied the phosphorylation of IκBα in the presence of inhibitor C3. Results showed a dose-dependent decrease of pIκB. So the next question was whether the phosphorylation of IKKβ was affected by nitrosylation. We did not detect any change in pIKKβ with different concentrations of C3. The decreased degradation of IκBα caused by C3 translated into decreased NF-κB activity as seen by a dose-dependent decrease in amounts of ICAM-1 with increasing C3 concentration. This data supports the premise that the activity of transcription factor NF-κB is suppressed by inhibiting GSNOR with compound C3

The nuclear factor-kB (NF-kB) signaling cascade is comprised of two branches, the canonical and alternative NF-kB pathways. Signaling through the alternative NF-kB pathway culminates in the activation of the downstream transcription factor subunits, RELB and NF-kB2. The biological roles of RELB and NF-kB2 within the B cell lineage have been obscured in constitutional knockout mice by the diverse functions of these subunits in non-B cell types. To overcome these limitations, conditional alleles were generated to investigate the roles of RELB and NF-kB2 in B cell development. These alleles allowed the identification of complex functional requirements for RELB and/or NF-kB2 in naïve B cells, germinal center (GC) B cells and plasma cells (PCs). These functional requirements may have implications for B cell malignancies that display mutations that constitutively activate the alternative NF-kB pathway. A large body of work has demonstrated that B cell activating factor (BAFF) signaling is critical for the maintenance of mature B cells. However, the contribution of the alternative NF-kB subunits that are activated downstream of BAFF remained unclear, especially in regards to their specific target genes. We have identified critical, B cell-intrinsic roles for RELB and NF-kB2 in the maintenance of mature B cells. In response to BAFF, these subunits were found to control the expression of anti-apoptotic genes, genes that ensure correct positioning within the B cell niche, and genes involved in promoting B–T cell interactions that allow effective antigen-mediated activation. During the GC B cell reaction, light zone (LZ) B cells undergo affinity-based selection mediated by T follicular helper (Tfh) cells. A subset of LZ B cells show activation of the NF-kB signaling cascade, suggesting a critical role for NF-kB in the selection of high-affinity GC B cells. We here report that GC B cell development occurred normally in mice with conditional deletion of either relb (RELB) or nfkb2 (NF-kB2) in GC B cells. In contrast, the simultaneous ablation of both subunits caused rapid involution of established GCs, similar to what has been observed for ablation of the canonical NF-kB transcription factor subunit c-REL. Intriguingly, RNA-sequencing analysis of relb/nfkb2-deleted GC B cells revealed no overlap between the genes controlled by RELB/p52 and c-REL within GC B cells. This suggests that signaling through the separate NF-kB pathways in GC B cells results in the expression of different biological programs that are independently required for the maintenance of the GC reaction. In addition, we observed that human PCs and PC precursors within the LZ showed high protein levels of NF-kB2 compared to surrounding lymphocytes, suggesting a biological role for this subunit in PCs. Indeed, ablation of nfkb2 alone in GC B cells led to a dramatic decrease in antigen-specific serum IgG1 and antigen-specific IgG1-secreting cells. Interestingly however, the mice developed normal frequencies of PCs, suggesting a role for NF-kB2 in PC physiology rather than differentiation.

TAL-1, ectopically expressed in 60% of T-cell acute lymphoblastic leukemia (T-ALL) patients, may contribute to poor chemotherapy response. This research sought to determine if TAL-1 influences expression of proteins involved in the NF-kB signaling pathway and thus, resistance to cell death. NF-kB, IKKy, and TRAF-2 expression levels were found to be TAL-1 dependent. Cell death levels were higher in staurosporine-treated cells compared to tumor necrosis factor a-treated or dual-treated cells. TAL-1, NF-kB, IKKy, and TRAF-2 expression levels were elevated in tumor necrosis factor a-treated cells and reduced in staurosporine-treated or dual treated cells compared to untreated cells. These results suggest TAL-1 influences expression of proteins involved in the NF-kB signaling pathway, thus inducing an anti-apoptotic response in the cell.<br>Department of Biology

Lou, Pak Kin.<br>Thesis (M.D.)--Chinese University of Hong Kong, 2011.<br>Includes bibliographical references (leaves 136-170).<br>Abstracts in English and Chinese.<br>Abstract --- p.i<br>Acknowledgements --- p.v<br>Table of Contents --- p.vi<br>List of Figures --- p.x<br>List of Tables --- p.xiii<br>List of Publications --- p.xv<br>Chapter Chapter 1 --- Introduction --- p.1<br>Chapter 1.1. --- Aims of Study --- p.1<br>Chapter 1.2. --- Literature Review --- p.2<br>Chapter 1.2.1. --- Nasopharyngeal Carcinoma --- p.2<br>Chapter 1.2.1.1. --- Overview --- p.2<br>Chapter 1.2.1.2. --- Histopathology --- p.2<br>Chapter 1.2.1.3. --- Epidemiology --- p.3<br>Chapter 1.2.1.4. --- Etiology --- p.5<br>Chapter 1.2.1.4.1. --- Epstein-Barr Virus (EBV) Latent Infection --- p.5<br>Chapter 1.2.1.4.2. --- Environmental Factors --- p.5<br>Chapter 1.2.1.4.3. --- Genetic Factors --- p.6<br>Chapter 1.2.1.5. --- Molecular Pathogenesis --- p.7<br>Chapter 1.2.1.5.1. --- Chromosomal Alterations --- p.7<br>Chapter 1.2.1.5.2. --- NPC-associated Tumour Suppressor Genes --- p.7<br>Chapter 1.2.1.5.3. --- NPC-associated Oncogenes --- p.8<br>Chapter 1.2.2. --- Epstein-Barr Virus --- p.9<br>Chapter 1.2.2.1. --- Overview --- p.9<br>Chapter 1.2.2.2. --- Lytic and Latent Infection of EBV --- p.9<br>Chapter 1.2.2.3. --- EBV Latency Programs and Associated --- p.10<br>Malignancies --- p.11<br>Chapter 1.2.2.4. --- The Role of EBV in NPC --- p.12<br>Chapter 1.2.3. --- NF-kB Signalling Pathways --- p.12<br>Chapter 1.2.3.1. --- Overview --- p.12<br>Chapter 1.2.3.2. --- Pathway Components --- p.12<br>Chapter 1.2.3.2.1. --- NF-kB Subunits --- p.16<br>Chapter 1.2.3.2.2. --- Inhibitors of kB (IkBs) --- p.16<br>Chapter 1.2.3.2.3. --- IkB Kinases (IKKs) --- p.17<br>Chapter 1.2.3.3. --- NF-kB Activation and Signalling --- p.17<br>Chapter 1.2.3.3.1. --- The Canonical Pathway --- p.18<br>Chapter 1.2.3.3.2. --- The Non-canonical Pathway --- p.18<br>Chapter 1.2.3.3.3. --- Physiological Functions of NF-kB --- p.19<br>Chapter 1.2.3.4. --- NF-kB Signalling and Tumourigenesis --- p.20<br>Chapter 1.2.3.4.1. --- Oncogenic Activation of NF-kB in Hematological Malignancies --- p.20<br>Chapter 1.2.3.4.2. --- Oncogenic Activation of NF-kB in Solid and Epithelial Tumours --- p.22<br>Chapter Chapter 2 --- Material and Methods --- p.22<br>Chapter 2.1. --- Tumour Specimens --- p.24<br>Chapter 2.2. --- NPC Tumour Lines and Immortalized NP Cell Lines --- p.24<br>Chapter 2.2.1. --- Cell Lines --- p.24<br>Chapter 2.2.2. --- Xenografts --- p.27<br>Chapter 2.3. --- DNA Sequence Analysis --- p.27<br>Chapter 2.3.1. --- Genomic DNA Extraction --- p.27<br>Chapter 2.3.2. --- Polymerase Chain Reaction (PCR) --- p.28<br>Chapter 2.3.3. --- DNA Sequencing --- p.32<br>Chapter 2.4. --- RNA Expression Analysis --- p.32<br>Chapter 2.4.1. --- Total RNA Extraction and Reverse Transcription --- p.33<br>Chapter 2.4.2. --- Quantitative Real-time Polymerase Chain Reaction (QRT-PCR) --- p.35<br>Chapter 2.5. --- Protein Expression Analysis --- p.35<br>Chapter 2.5.1. --- Total Protein Extraction --- p.35<br>Chapter 2.5.2. --- Nuclear and Cytoplasmic Protein Isolation --- p.36<br>Chapter 2.5.3. --- Western Blotting --- p.39<br>Chapter 2.6. --- Immunohistochemical Staining --- p.41<br>Chapter 2.7. --- Statistical Analysis --- p.41<br>Chapter 2.8. --- Immunoprecipitation --- p.43<br>Chapter 2.9. --- Electrophoretic Mobility Shift Assay (EMSA) and Supershift Assay --- p.44<br>Chapter 2.10. --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.45<br>Chapter 2.11. --- Plasmid Preparation --- p.45<br>Chapter 2.11.1. --- Plasmids --- p.45<br>Chapter 2.11.2. --- Bacterial Transformation and Plasmid DNA Extraction --- p.46<br>Chapter 2.12. --- Transfections --- p.46<br>Chapter 2.12.1. --- Transient Transfection --- p.46<br>Chapter 2.12.2. --- Stable Transfection --- p.47<br>Chapter 2.13. --- Immunofluorescence --- p.47<br>Chapter 2.14. --- Cell Proliferation and Viability Analysis --- p.47<br>Chapter 2.15. --- Small Interfering RNA (siRNA) Knockdown --- p.49<br>Chapter 2.16. --- Expression Microarray --- p.49<br>Chapter 2.16.1. --- Agilent Oligonucleotide Microarray --- p.50<br>Chapter 2.16.2. --- Data Analysis --- p.51<br>Chapter Chapter 3 --- Activation of NF-kB Signals in NPC --- p.51<br>Chapter 3.1. --- Introduction --- p.52<br>Chapter 3.2. --- Results --- p.52<br>Chapter 3.2.1. --- Expression Pattern of NF-kB Subunits in NPC Tumour Lines --- p.55<br>Chapter 3.2.2. --- Distinct NF-kB Complexes in NPC Tumour Lines --- p.60<br>Chapter 3.2.3. --- Expression of NF-kB Subunits in NPC Primary Tumours --- p.67<br>Chapter 3.3. --- Discussion<br>Chapter Chapter 4 --- Alterations of NF-kB Components in NPC --- p.71<br>Chapter 4.1. --- Introduction --- p.72<br>Chapter 4.2. --- Results --- p.72<br>Chapter 4.2.1. --- Homozygous Deletion of IicBa and TRAF3 in NPC Tumour Lines --- p.76<br>Chapter 4.2.2. --- Mutation of TRAF2 and A20 in NPC Tumour Lines<br>Chapter 4.2.3. --- Aberrant Expression of Multiple NF-kB Signalling Components in NPC Tumour Lines --- p.80<br>Chapter 4.2.4. --- Expression of NF-kB Signalling Components in NPC --- p.85<br>Primary Tumour --- p.92<br>Chapter 4.3. --- Discussion --- p.99<br>Chapter Chapter 5 --- Identification of Downstream Targets for NPC-associated NF-kB Signalling --- p.99<br>Chapter 0.1. --- Introduction --- p.99<br>Chapter 0.2. --- Results --- p.100<br>Chapter 0.2.1. --- Target Genes Modulated by p50 --- p.100<br>Chapter 0.2.2. --- Functional Annotation of p50 Target Genes --- p.105<br>Chapter 0.2.3. --- Target Genes Modulated by RelB --- p.105<br>Chapter 0.2.4. --- Functional Annotation of RelB Target Genes --- p.105<br>Chapter 0.2.5. --- Functional Annotation of Genes Modulated by both p50 and RelB --- p.111<br>Chapter 0.3. --- Discussion --- p.118<br>Chapter Chapter 6 --- Functional Role of TRAF3 Inactivation in NPC --- p.118<br>Chapter 0.1. --- Introduction --- p.118<br>Chapter 0.2. --- Results --- p.118<br>Chapter 0.2.1. --- Effect of TRAF3 Restoration on NF-kB Activity --- p.119<br>Chapter 0.2.2. --- Effect of TRAF3 Expression on Cell Proliferation --- p.123<br>Chapter 0.2.3. --- TRAF3 Expression Modulates Interferon Transcription in NPC Cells --- p.128<br>Chapter 0.3. --- Discussion<br>Chapter Chapter 7 --- General Discussion --- p.132<br>Chapter Chapter 8 --- Conclusion<br>Chapter Chapter 9 --- References<br>Appendix --- p.136

OBJECTIVE: Development of treatment resistance and adverse toxicity associated with classical chemotherapeutic agents highlights the need for safer and effective therapeutic approaches. Herein, we examined the effectiveness of a combination treatment regimen of 5-fluorouracil (5-FU) and curcumin in colorectal cancer (CRC) cells. METHODS: Wild type HCT116 cells and HCT116+ch3 cells (complemented with chromosome 3) were treated with curcumin and 5-FU in a time- and dose-dependent manner and evaluated by cell proliferation assays, DAPI staining, transmission electron microscopy, cell cycle analysis and immunoblotting for key signaling proteins. RESULTS: The individual IC50 of curcumin and 5-FU were approximately 20 microM and 5 microM in HCT116 cells and 5 microM and 1 microM in HCT116+ch3 cells, respectively (p<0.05). Pretreatment with curcumin significantly reduced survival in both cells; HCT116+ch3 cells were considerably more sensitive to treatment with curcumin and/or 5-FU than wild-type HCT116 cells. The IC50 values for combination treatment were approximately 5 microM and 1 microM in HCT116 and 5 microM and 0.1 microM in HCT116+ch3, respectively (p<0.05). Curcumin induced apoptosis in both cells by inducing mitochondrial degeneration and cytochrome c release. Cell cycle analysis revealed that the anti-proliferative effect of curcumin and/or 5-FU was preceded by accumulation of CRC cells in the S cell cycle phase and induction of apoptosis. Curcumin potentiated 5-FU-induced expression or cleavage of pro-apoptotic proteins (caspase-8, -9, -3, PARP and Bax), and down-regulated anti-apoptotic (Bcl-xL) and proliferative (cyclin D1) proteins. Although 5-FU activated NF-kappaB/PI-3K/Src pathway in CRC cells, this was down-regulated by curcumin treatment through inhibition of IkappaBalpha kinase activation and IkappaBalpha phosphorylation. CONCLUSIONS: Combining curcumin with conventional chemotherapeutic agents such as 5-FU could provide more effective treatment strategies against chemoresistant colon cancer cells. The mechanisms involved may be mediated via NF-kappaB/PI-3K/Src pathways and NF-kappaB regulated gene products.

Resveratrol, an activator of histone deacetylase Sirt-1, has been proposed to have beneficial health effects due to its antioxidant and anti-inflammatory properties. However, the mechanisms underlying the anti-inflammatory effects of resveratrol and the intracellular signaling pathways involved are poorly understood. An in vitro model of human tenocytes was used to examine the mechanism of resveratrol action on IL-1beta-mediated inflammatory signaling. Resveratrol suppressed IL-1beta-induced activation of NF-kappaB and PI3K in a dose- and time-dependent manner. Treatment with resveratrol enhanced the production of matrix components collagen types I and III, tenomodulin, and tenogenic transcription factor scleraxis, whereas it inhibited gene products involved in inflammation and apoptosis. IL-1beta-induced NF-kappaB and PI3K activation was inhibited by resveratrol or the inhibitors of PI3K (wortmannin), c-Src (PP1), and Akt (SH-5) through inhibition of IkappaB kinase, IkappaBalpha phosphorylation, and inhibition of nuclear translocation of NF-kappaB, suggesting that PI3K signaling pathway may be one of the signaling pathways inhibited by resveratrol to abrogate NF-kappaB activation. Inhibition of PI3K by wortmannin attenuated IL-1beta-induced Akt and p65 acetylation, suggesting that p65 is a downstream component of PI3K/Akt in these responses. The modulatory effects of resveratrol on IL-1beta-induced activation of NF-kappaB and PI3K were found to be mediated at least in part by the association between Sirt-1 and scleraxis and deacetylation of NF-kappaB and PI3K. Overall, these results demonstrate that activated Sirt-1 plays an essential role in the anti-inflammatory effects of resveratrol and this may be mediated at least in part through inhibition/deacetylation of PI3K and NF-kappaB.