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Amin, Jakia, Ken-ichiro Otsuyama, Abul Islam, Karim Shamsasenjan, Shamim Mohd Iqbal, and Michio M. Kawano. "The Mechanism of Constitutive NF-kB Activity in Myeloma Cell Lines." Blood 110, no. 11 (November 16, 2007): 4740. http://dx.doi.org/10.1182/blood.v110.11.4740.4740.
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Abstract [Purpose] NF-kB has a key function in the transformation, proliferation and invasion of cancer cells as well as in resistance to radiotherapy and chemotherapy. Although constitutive NF-kB activation has been reported in many human tumors, the underlying factors and mechanisms responsible for constitutive NF-kB activation in myeloma cells has not been known. The purpose of this study was to determine the mechanism of constitutive NF-kB activity in myeloma cell lines and quantification of NF-kB activity in primary myeloma cells by expression of CD54 (a NF-kB target gene). [Methods] We checked the constitutive expression of NF-kB family proteins by western blot analysis and possible dimer formation of different NF-kB family members by Immunoprecipitation-western blot reaction. Constitutive NF-kB DNA-binding activity and dimers that are responsible for NF-kB activity were analyzed by electrophoretic mobility shift assay (EMSA). Moreover, expression of different NF-kB target genes was done by RT-PCR. [Results and discussion] Constitutive NF-kB activity was determined in myeloma cell lines and our results suggested that p50/RelB, p50/p50, p50/p52 dimers might be responsible for this. We also analyzed several NF-kB target gene expressions and found that the intensity of CD54 expression was positively correlated with total NF-kB DNA binding activity. Although there is no method to quantified NF-kB activity, it can be determine in terms of its target genes e.g. CD54. Therefore, this study provide the frame work for understanding the molecular mechanism of constitutive activation of NF-kB and would help to quantify NF-kB activation in primary myeloma cells.
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Ghosh, Sankar, Eijiro Jimi, Jie Dong, and Haihong Zhong. "REGULATION OF NF-KB TRANSCRIPTIONAL ACTIVITY." Shock 21, Supplement (March 2004): 44. http://dx.doi.org/10.1097/00024382-200403001-00176.
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Otsuyama, Ken-ichiro, Jakia Amin, Saeid Abroun, Abul Islam, Karim Shamsasenjan, Shamim Mohd Iqbal, and Michio M. Kawano. "The PPARß Activation Mechanism Which Suppresses the Constitutive NF-kB Activity in Human Myeloma Cell Lines." Blood 110, no. 11 (November 16, 2007): 4739. http://dx.doi.org/10.1182/blood.v110.11.4739.4739.
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Abstract [Purpose] It is considered that human myeloma cells have the constitutively high NF-kB activity involved in survival and proliferation. PPAR (Peroxisome proliferator-activated receptor) ß is ubiquitously expressed in all cells and considered to be involved in the lipid metabolism and regulating the inflammatory response and cell proliferation. We already have found that adrenal cortex hormones (DHEA and DHEA-S etc), dexamethasone (Dex) and baicalein augmented the expression of reporter gene. However, it remain to be clarified the role of PPARß in human myeloma cells. We focus on the mechanism of PPARß suppressed NF-kB activity. [Method] To know NF-kB activity of human myeloma cells, we performed EMSA with NF-kB consensus oligo. To investigate NF-kB and PPARß after stimulation of PPAR agonist, we did EMSA with NF-kB and PPAR consensus oligo. To confirm whether it is repression according the NF-kB activity suppression accompanying PPARß activation to the interaction of PPARß and NF-kB, we conducted immunoprecipitation - western blot analysis. To check whether the expression of NF-kB target genes (cIAP1, Bcl-xL, etc) were suppressed after stimulation of PPAR agonist, we performed RT-PCR analysis. [Result and discussion] It was suggested that human myeloma cell lines have constitutive NF-kB activity, and its activity mainly regulated p50. NF-kB activity and its target genes were repressed by stimulation of PPAR agonist. From the above, it was suggested that the activated PPARß interacted NF-kB and then its activity was suppressed.
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Magrangeas, Florence, Philippe Moreau, Loic Campion, Herve Avet-Loiseau, Catherine Guérin, Wilfried Gouraud, Gérald Marit, et al. "Low Level Of NF-Kb Activity Is Associated With Higher Response Rate To Bortezomib-Based Induction Therapy In Patients With Newly Diagnosed Multiple Myeloma." Blood 122, no. 21 (November 15, 2013): 3106. http://dx.doi.org/10.1182/blood.v122.21.3106.3106.
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Abstract Purpose Activation of canonical and noncanonical NF-kB pathways plays a key role in multiple myeloma (MM) pathogenesis. Recent studies have shown that constitutive activation of NF-kB pathways is present in ∼15-20 % of newly diagnosed MM patients. Bortezomib is a potent selective inhibitor of NF-kB activation; however its cytotoxicity is mainly due to inhibition of the noncanonical NF-kB pathway. We have previously shown that the achievement of very good partial response (VGPR) after induction therapy prior to frontline autologous stem cell transplantation (ASCT) was a favourable prognostic factor for progression-free survival (Moreau et al, Blood 2011;117:3041-3044). Our aim was to correlate NF-kB activity, reflected by a gene signature of well-known NF-kB targets, with the response rate achieved with a bortezomib-based induction regimen prior to high-dose therapy and ASCT performed as part of frontline treatment in patients with symptomatic multiple myeloma. Patients and methods One hundred and ninety-nine patients with symptomatic MM were enrolled in the prospective randomized IFM2007-02 trial comparing 4 cycles of bortezomib-dexamethasone (VD) induction therapy versus 4 cycles of the triplet combination bortezomib-thalidomide-dexamethasone (VTD) prior to ASCT. Among these 199 patients, 114 included in the present study were available for gene expression profile testing and therefore analysed for NF-kB activity. For each patient, the NF-kB(10) index, a reliable measure of NF-kB activity in MM tumor cells (Demchenko et al, Blood 2010; 115: 3541-3552) was calculated. NF-kB(10) index is based on a transcription signature of 10 genes: IL2RG, NFKB2,TNFAIP3, NFKBIE, NFKBIA, RELB, CD74, PLEK, MALT1 and WNT10. Gene expression signature was obtained from Affymetrix Exon1.0 normalized data. A high level of NF-kB activity was defined by the NF-kB(10) index found in a cohort of 20 MM patients with biallelic deletions, identified by using Affymetrix SNP6.0 data, that inactivate negative regulators (cIAP1/2) of NF-kB pathways. Response to induction therapy was evaluated according to the IMWG criteria. Results Responses to induction therapy are shown in Table 1. In this subgroup analysis, the CR plus VGPR rates were not statistically different in both arms of the trial: 24 / 54 (44%) in the VTD arm versus 21 / 60 (35%) in the VD arm, P = .35. We subsequently analyzed the correlation between NF-kB activity and response in the whole cohort of 114 patients regardless of induction treatment. We found that the level of NF-kB activity, based NF-kB(10) index, tested as a continuous variable, was strongly correlated with the quality of response, i.e. VGPR or better (P = .007 Wilcoxon test). We also investigated the cut-off value of NF-kB(10) index that could impact the response rate. Since high NF-kB(10) index was found in MM cells with known NF-kB mutations, we calculated the NF-kB(10) index in a control cohort of MM patients deleted in cIAP1/2. Patients of IFM2007-02 trial with a NF-kB(10) index (< 70.5) lower than the index of the control cohort were assigned to the low NF-kB activity group (50/114, 44%); therefore the remaining cases (64/114, 56%) presented with a high NF-kB activity. We found that patients with a reduced NF-kB(10) index displayed a significantly higher response rate (27/50; 54% vs 18/64; 28%, P= .007), as shown in Figure 1, indicating that the vast majority of patients with high NF-kB were not able to achieve at least VGPR. Conclusion Our results show that a low level NF-kB activity is associated with a higher response rate (>VGPR) to bortezomib-based induction regimen. Patients with low NF-kB(10) index represent 44% of the cohort studied. Since NK-kB activity is related to both canonical and noncanonical pathways, and knowing that bortezomib-induced cytotoxicity is mostly due to the inhibition of the noncanonical pathway only, our data strongly suggest that bortezomib should be combined with drug also targeting the canonical pathway in order to induce a high response rate in patients with increased NF-kB activity, as shown in preclinical studies (Fabre et al, Clin Cancer Res. 2012;18:4669-4681. Disclosures: Moreau: CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau. Off Label Use: FRONTLINE TREATMENT WITH CARFIZOMIB. Attal:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau. Facon:Janssen and Celgene: Speakers Bureau; Millennium, Onyx, Novartis, BMS, Amgen: Membership on an entity's Board of Directors or advisory committees.
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Tsai, Hui-Jen, Seiichiro Kobayashi, Kiyoko Itoh, Takaomi Ishida, Kazuo Umezawa, and Arinobu Tojo. "Microenvironmental Up-Regulation of NF-kB Activity Via P65-Dependent and Independent Pathways in a Bioimaging Model of Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia." Blood 110, no. 11 (November 16, 2007): 723. http://dx.doi.org/10.1182/blood.v110.11.723.723.
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Abstract Constitutive activation of NF-kB via Bcr-Abl has been demonstrated in primary blast cells and cell lines derived from Philadelphia chromosome (Ph) -positive acute lymphoblastic leukemia (Ph-ALL). However, the microenvironmental (cytokine and/or stroma cell) regulation of NF-kB activity in Ph-ALL has not been clarified. To gain insight into these unsolved issues, we lentivirally transduced IMS-PhL1 cells with NF-kB/luciferase (kB/Luc) reporter construct and established a bioluminescence imaging model of Ph-ALL for in vitro and in vivo analysis. Unstimulated PhL1-kB/Luc cells revealed a weak but significant Luc activity over the background, verifying constitutive activation of NF-kB. Among a panel of cytokines, only TNFa potently up-regulated Luc activity in PhL1-kB/Luc cells about 10-fold over the basal level. DHMEQ, a specific inhibitor of nuclear translocation of p65, eradicated constitutive and TNFa-inducible NF-kB activity of PhL1 cells and induced their substantial apoptosis dose-dependently. A series of Ph-ALL cell lines were similarly sensitive to treatment with DHMEQ, suggesting the critical role of constitutive NF-kB activity in survival of Ph-ALL cells. When PhL1-kB/Luc cells were seeded onto a layer of murine HESS-5 stroma cells, Luc activity was not changed. Intriguingly, TNFa stimulation of PhL1-kB/Luc cells in the presence of HESS-5 cells caused synergistic enhancement of Luc activity up to 20 fold over the basal level. This up-regulation was canceled by blocking cell to cell contact with a transwell membrane, suggesting that the direct cell contact may be essential for such a synergistic effect. In HESS-5 cells, NF-kB activity was markedly augmented in response to TNFa, but this up-regulation was not sensitive to DHMEQ. Furthermore, the inhibitory effects of DHMEQ on Luc activity as well as viability of TNFa-treated PhL1-kB/Luc cells were significantly alleviated in the presence of HESS-5 cells. (Fig 1) Taken together, TNFa-triggered HESS-5 cells are likely to up-regulate NF-kB activity of PhL1 cells through DHMEQ-insensitive alternate pathway. Finally, PhL1-kB/Luc cells were transplanted into NOD-SCID mice and subjected to periodic monitoring with a CCD camera. (Fig 2) We successfully detected constitutive and TNFa-inducible bioluminescent signals of leukemia cells. Unexpectedly, by far the strongest constitutive signal was captured in the liver, although massive leukemic infiltration was observed in bone marrow and spleen, implying that hepatic microenvironment may offer proper stimuli to activate NF-kB and constitute leukemic niche. In conclusion, p65-dependent and independent pathways are involved in microenvironmental up-regulation of NF-kB activity, which contribute to survival, expansion and presumably drug-resistance of Ph-ALL cells. The present bioimaging model helps us to dissect the regulatory mechanism of NF-kB signal by cytokines and cellular interactions. Fig. 1 Up-regulation of NF-κB activity in Ph-ALL cells by cytokines and/or stroma. Fig. 2 In vivo imaging of NF-κB activity in Ph-ALL cells Fig. 1. Up-regulation of NF-κB activity in Ph-ALL cells by cytokines and/or stroma. . / Fig. 2 In vivo imaging of NF-κB activity in Ph-ALL cells
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Bao, Xiaoyong, Deepthi Kolli, Tianshuang Liu, Yichu Shan, Roberto P. Garofalo, and Antonella Casola. "Human Metapneumovirus Small Hydrophobic Protein Inhibits NF-κB Transcriptional Activity." Journal of Virology 82, no. 16 (June 11, 2008): 8224–29. http://dx.doi.org/10.1128/jvi.02584-07.
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ABSTRACT Human metapneumovirus, a leading cause of respiratory tract infections in infants, encodes a small hydrophobic (SH) protein of unknown function. In this study, we showed that infection of airway epithelial cells or mice with recombinant human metapneumovirus lacking SH expression (rhMPV-ΔSH) enhanced secretion of proinflammatory mediators, including interleukin 6 (IL-6) and IL-8, encoded by two NF-kB-dependent genes, compared to infection with wild-type rhMPV. RhMPV-ΔSH infection resulted in enhanced NF-kB-dependent gene transcription and in increased levels of phosphorylated and acetylated NF-kB without affecting its nuclear translocation, identifying a possible novel mechanism by which paramyxovirus SH proteins modulate NF-kB activation.
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Wang, Xianhuo, Huaqing Wang, Chengfeng Bi, Xiaoyan Zhang, Xin Huang, Xuan Zhang, Javeed Iqbal, et al. "Microrna-17~92 Cluster Upregulates NF-KB Activity Via Suppressing Multiple NF-KB Negative Regulators Mediating Ubiquitination." Blood 126, no. 23 (December 3, 2015): 3638. http://dx.doi.org/10.1182/blood.v126.23.3638.3638.
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Abstract The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is a very aggressive lymphoma characterized by constitutive NF-kB activation, but whether miRNAs dysfunction contributes to this event, and their exact function and mechanism remain unclear. Starting from an integrative screening strategy, we revealed that there were some interactions between the NF-kB signaling and miR-17~92 cluster, which was essential for B-cell development and commonly gained and/or overexpressed in ABC-DLBCL. Several important NF-kB negative regulators including TNFAIP3 (A20), CYLD and Rnf11 were predicted and validated to be the direct targets of miR-17~92. Conditional knock-down of miR-17~92 using sponge could suppress NF-kB activity and elevate the A20, CYLD and Rnf11 expression in 293T cells. Furthermore, we demonstrated that enforced overexpression of miR-17~92 could also decrease the A20, CYLD and Rnf11 expression in ABC-DLBCL cells. Conditional overexpression of miR-17~92 could promote ABC-DLBCL cells growth, accelerate the cells G1/G0 phase to S phase transition, and suppress NF-kB inhibitor-induced apoptosis. Conversely, conditional knock-down of miR-17~92 could inhibit ABC-DLBCL cells growth and sensitize the cells to NF-kB inhibitor-induced apoptosis. The miR-17~92 could induce the IkB-a and NF-kB p65 phosphorylation, leading to the NF-kB activation and aberrant expression of NF-kB transcriptional target genes. However, miR-17~92 did not regulate the NF-kB p52/p100 phosphorylation. Overexpression of miR-17~92 enhanced K63-linked ubiquitination and reduced K48-linked ubiquitination of the TNFa receptor 1 complex including RIP1. Importantly, we found that high expression level of miR-17~92 was associated with poorer survival in ABC-DLBCL patients. Our results uncovered a novel mechanism for the canonical but not the non-canonical transcription factor NF-kB pathway by modulation of miR-17~92 in ABC-DLBCL, and suggested that targeting the miR-17~92 might be novel bio-therapeutic strategies, which could be single-agent or combined with NF-kB inhibitor treatment, for ABC-DLBCL patients. Figure 1. Inhibition of miR-17~92 blocks the activity of NF-kB in HER293T cells: (A) The schematic representation of reporter constructs involved in assay. (B) HEK293T cells were co-transfected with Dul-Luciferase reporter constructs and the miR-17~92 sponge plasmid. TNF-a stimulation or without stimulation for 18 h. Figure 1. Inhibition of miR-17~92 blocks the activity of NF-kB in HER293T cells: (A) The schematic representation of reporter constructs involved in assay. (B) HEK293T cells were co-transfected with Dul-Luciferase reporter constructs and the miR-17~92 sponge plasmid. TNF-a stimulation or without stimulation for 18 h. Figure 2. miR-17~92 directly regulates A20, CYLD and Rnf11 in ABC-DLBCL cells. (A) Fluorescence images of tranduced ABC-DLBCL cells. (B) Expression of sponge or miR-17~92 in tranduced ABC-DLBCL cells. (C) Inhibition of miR-17~92 increase the expression of A20, CYLD and Rnf11. Overexpression of miR-17~92 reduce the expression of A20, CYLD and Rnf11 in ABC-DLBCL cells. Figure 2. miR-17~92 directly regulates A20, CYLD and Rnf11 in ABC-DLBCL cells. (A) Fluorescence images of tranduced ABC-DLBCL cells. (B) Expression of sponge or miR-17~92 in tranduced ABC-DLBCL cells. (C) Inhibition of miR-17~92 increase the expression of A20, CYLD and Rnf11. Overexpression of miR-17~92 reduce the expression of A20, CYLD and Rnf11 in ABC-DLBCL cells. Figure 3. miR-17~92 modulate mediate NF-kB activity in ABC-DLBCL. (A) Immunoblot analysis of IkB-a, NF-kB p65, NF-kB p100/p52 and their phosphorylation. (B) Heat-map display of quantitative real-time RT-PCR measurements of six independent NF-kB transcriptional targets show significantly lower expression in sponge expressing cells and higher expression in miR-17~92 expressing cells. Figure 3. miR-17~92 modulate mediate NF-kB activity in ABC-DLBCL. (A) Immunoblot analysis of IkB-a, NF-kB p65, NF-kB p100/p52 and their phosphorylation. (B) Heat-map display of quantitative real-time RT-PCR measurements of six independent NF-kB transcriptional targets show significantly lower expression in sponge expressing cells and higher expression in miR-17~92 expressing cells. Disclosures No relevant conflicts of interest to declare.
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Vassiliev, P. M., A. A. Spasov, L. R. Yanaliyeva, A. N. Kochetkov, V. V. Vorfolomeyeva, V. G. Klochkov, and D. T. Appazova. "Neural network modeling of multitarget RAGE inhibitory activity." Biomeditsinskaya Khimiya 65, no. 2 (2019): 91–98. http://dx.doi.org/10.18097/pbmc20196502091.
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Based on the methodology of artificial neural networks, models describing the dependence of the level of RAGE inhibitory activity on the affinity of compounds for target proteins of the RAGE-NF-kB signal pathway have been costructed. A validated database of the structures and activity levels of 183 known compounds, which were tested for RAGE inhibitory activity was formed. The analysis of the AGE-RAGE signaling pathways was carried out, 14 key RAGE-NF-kB signal pathway nodes were found, for which 34 relevant target proteins were identified. A database of 66 valid 3D models of 22 target proteins of the RAGE-NF-kB signal chain was compiled. Ensemble molecular docking of 3D models of 183 known RAGE inhibitors into sites of 66 valid 3D models of 22 relevant RAGE target proteins was performed and minimum docking energies for each compound were determined for each target. According to the method of artificial multilayer perceptron neural networks, classification models were constructed to predict level of RAGE inhibitory activity based on the calculated affinity of compounds for significant target proteins of the RAGE-NF-kB signaling chain. The prognostic ability of these models of RAGE-inhibitory activity was evaluated, the maximum accuracy according to ROC-analysis was 90% for a high level of activity. The sensitivity analysis of the developed multitarget models were carried out, the most significant targets of the RAGE-NF-kB signal transmission chain were determined. It was found that for high level of RAGE inhibitory activity, the most significant biotargets are not AGE receptors, but eight signaling kinases of the RAGE-NF-kB pathway and transcription factor NF-kB1. Thus, it is suggested that known compounds with high RAGE-inhibitory activity are preferential inhibitors of signal kinases.
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Neumann, Manfred, and Michael Naumann. "Beyond IκBs: alternative regulation of NF‐KB activity." FASEB Journal 21, no. 11 (April 12, 2007): 2642–54. http://dx.doi.org/10.1096/fj.06-7615rev.
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Nakagawa, Masahiro, Munetake Shimabe, Nahoko Nishimoto, Naoko Watanabe-Okochi, Motoshi Ichikawa, Yasuhito Nannya, Yoichi Imai, and Mineo Kurokawa. "AML1/Runx1 Is a Cytoplasmic Attenuator of NF-Kb Signaling: Implication in Pathogenesis and Targeted Therapy of AML1-Related Leukemia." Blood 114, no. 22 (November 20, 2009): 1962. http://dx.doi.org/10.1182/blood.v114.22.1962.1962.
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Abstract Abstract 1962 Poster Board I-985 Introduction: AML1/Runx1 is one of the most frequent targets of chromosomal abnormalities in human leukemia. Functional impairment of AML1 caused by point mutation is also reported in patients with leukemia or myelodysplastic syndrome (MDS). However, molecular basis for leukemogenesis caused by functional impairment of AML1 is still elusive. In this study, we clarified the deregulated signaling pathway induced by loss of AML1. Results: To find the direct target of AML1, we compared gene expression profile between AML1-conditionally deleted and normal KSL cells using Cre-ER system. Gene set enrichment analysis (GSEA) using molecular signature database (MSigDB) clarified enhanced expression of NF-kB target genes in AML1 deficient cells. In addition, NF-kB inhibitor attenuated the enhanced colony forming activity of bone marrow cells from AML1 conditional knockout (cKO) mice. These data indicate the aberrant activation of NF-kB signaling pathway in stem/progenitor cells of AML1 deficient mice. NF-kB is a transcription factor which is involved in many physiological phenomena including proliferation, survival, and inflammation. Because deregulated activation of NF-kB signaling has been reported to be responsible for many types of tumors including hematological malignancies, we assumed that lack of AML1-mediated suppression of NF-kB signaling lead to malignant transformation of hematopoietic cells. p65, one of the major components of NF-kB stays in cytoplasm with IkB in a steady state. Once receiving stimulating signals from cell surface receptors such as TNF-a receptor, IkB is phosphorylated by IKK complex and subsequently degraded through the ubiquitin-proteasome pathway, resulting in nuclear translocation of p65 and transactivation of NF-kB target genes. First, we found that AML1 inhibits nuclear translocation of p65 and that nuclear localization of p65 is enhanced in AML1 deficient cells, which is cancelled by NF-kB inhibitors. In addition, AML1 inhibited p65 phosphorylation at serine 536, which is important for its activation. We found that AML1 physically interacts with IKK complex and thus suppresses its kinase activity, which accounts for a mechanistic basis for inhibition of NF-kB signaling by AML1. Suppression of IKK kinase activity by AML1 results in inhibition of both nuclear translocation of p65 and activation of NF-kB target genes. Next, we examined how leukemia-related AML1 mutants affect NF-kB signaling. Remarkably, AML1 D171N mutant found in MDS neither inhibited nuclear translocation of p65 nor attenuated the kinase activity of IKK complex. Similar results were obtained with AML1/ETO generated in leukemia with t(8;21). Mouse bone marrow cells immortalized by AML1/ETO showed enhanced nuclear localization of p65 compared with those immortalized by MLL/ENL, another leukemia-related fusion protein. Indeed, AML1/ETO immortalized cells are more sensitive to NF-kB inhibitor-mediated growth suppression, indicating a critical role of NF-kB signaling in transformation by AML1/ETO. To verify the activation of NF-kB signaling by AML1/ETO in human hematopoietic cells, we analyzed the gene expression data reported by Valk et al. in silico. We found that NF-kB signaling is distinctly activated in AML1-related leukemia patients. These results suggest that aberrant activation of NF-kB signaling induced by functional impairment of AML1 may contribute to the development of leukemia via proliferation signals. Conclusions: We found that AML1 is a cytoplasmic attenuator of NF-kB signaling pathway. Functional impairment of AML1 caused by genetic disruption results in distinct activation of NF-kB signaling by altering IKK kinetic activity. This aberrant activation may play a central role in pathogenesis of AML1-related leukemia and MDS. Therefore, NF-kB signaling is one of the attractive candidates for molecular targeted therapy against AML1-related hematological disorders. Disclosures: No relevant conflicts of interest to declare.
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Hussain, Sajjad, and Paul J. Galardy. "UCH-L1 Provides An Essential Survival Signal in Malignant B-Cells by Enhancing NF-κB Activation." Blood 112, no. 11 (November 16, 2008): 3338. http://dx.doi.org/10.1182/blood.v112.11.3338.3338.
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Abstract Using an activity-based approach, we previously described the over-expression of the deubiquitinase ubiquitin carboxy terminal hydroxylase 1 (UCH-L1) in several B-cell malignancies including multiple myeloma, Burkitt lymphoma, non-Hodgkin lymphoma and chronic lymphocytic leukemia. UCH-L1 over expression also correlates with a poor prognosis in non-lymphoid tumors including non-small cell lung, esophageal, colorectal and pancreatic cancers. Whether UCH-L1 expression contributes to the malignant phenotype in these conditions, however, has not been established. We therefore sought to determine the role of UCH-L1 in lymphoid malignancies through the use of lentivirusencoded short hairpin RNA (shRNA) targeting UCH-L1. When compared with a control non-silencing shRNA, reduced UCH-L1 levels are associated with a profound block in proliferation and loss of viability (>90%) in the UCH-L1 expressing myeloma cell line KMS11. The introduction of these shRNAs into the UCH-L1 negative myeloma cell line KMS12 does not affect proliferation or viability. The association of cell death with reduced UCH-L1 levels was further reinforced using a doxycycline inducible shRNA construct. KMS11 and KMS12 cells grow equally well following transduction and selection of stable viral integrants. However, upon induction of shRNA production with doxycycline, greater than 90% of KMS11 cells die within 48-hours timed with the depletion of UCH-L1 while KMS12 cells continue to proliferate. These results strongly suggest that UCH-L1 is essential for survival in UCH-L1 expressing myeloma cells. Given the critical role of NF-kB signaling in many lymphoid malignancies, we examined the impact of UCH-L1 depletion on NF-kB activity. Using a secreted luciferase reporter assay, we determined NF-kB activity in control and UCH-L1 depleted KMS11 cells. We observed a 5-fold reduction of NF-kB activity in UCH-L1 knockdown cells compared to control. In order to establish a correlation between loss of UCH-L1 expression and NF-kB down-regulation, cells transduced with an inducible shRNA were treated with doxycycline and monitored daily for UCH-L1 and NF-kB activation. We observed a direct relationship between the level of UCH-L1 and NF-kB activity in these cells with a 5-fold reduction in NF-kB activity by 48-hours accompanied by reduced viability. These effects are entirely reversible with the removal of doxycycline from the growth medium. Doxycycline induction had no effect on the NF-kB activity of the UCH-L1 negative cell line KMS12. Knockdown of UCH-L1 does not affect TNF-alpha-induced NF-kB activity in these cells suggesting that UCH-L1 modulates the non-canonical NF-kB pathway. A relationship between UCH-L1 and NF-kB is further suggested by observing a 3.8-fold increase in baseline NF-kB activity in HeLa cells over-expressing UCH-L1. These data strongly indicate that UCH-L1 enhances NF-kB through the non-canonical pathway. In order to determine whether UCH-L1 over-expression can by itself promote tumorigenesis in vivo, we have engineered a UCH-L1 transgenic mouse. These mice will provide us the opportunity to study the effect of UCH-L1 expression on spontaneous as well as induced (carcinogen or oncogene) tumorigenesis. Further studies are examining the effect of forced UCH-L1 expression on B-cell development. This study will provide a better understanding of the role of UCH-L1 in hematological malignancies and may validate UCH-L1 as an important drug target for B-cell malignancies.
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Guo, Feng, Peng Zhou, and Liang Ma. "TRAF3 Over-Expression Contributes to the Enhanced Alternative NF-κB Activity in Hodgkin's Lymphoma Cells." Blood 114, no. 22 (November 20, 2009): 3665. http://dx.doi.org/10.1182/blood.v114.22.3665.3665.
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Abstract Abstract 3665 Poster Board III-601 Introduction Hodgkin and Reed-Sternberg (H-RS) cells are originated from germinal center B cells. Constitutive nuclear factor κB (NF-κB) activation is one of the molecular characteristic futures of H-RS cells. TNFR-associated factors (TRAFs) participate in a wide range of biological processes, such as adaptive and innate immunity, stress response, and bone metabolism, which are mediated by the induction of cell survival, proliferation, and differentiation. Among those, TRAF3 are reported as a negative regulator of the alternative NF-κB signaling pathway in B cells. How TRAF3 functions in H-RS cells is currently unclear. Methods Electromobility shift assay (EMSA) was performed to examine the NF-κB activity in B cell-derived Hodgkin's cells (L428 and KM-H2). An ELISA-based NF-κB family transcription factor activity assay was performed to quantify NF-κB DNA-binding in nuclear extracts from L428 cells. p100 processing, the expression of other NF-κB family members in the cytoplasm, and TRAF3 expression were detected by Western blot analysis. The effects of TRAF3 in L428 cells were studied by transient expression of TRAF3 expression vector. Results In this study, we found that TRAF3 was minimally detected in B cell-derived Hodgkin's cell lines (L428 and KM-H2) either in mRNA or protein levels. Both the classical (p50-RelA) and the alternative (p52-RelB) NF-kB activity were consistently activated in L428 cells, measured by EMSA and TransAM NF-kB activity assay. The enhanced alternative NF-κB activity, accompanied by increased p100 processing and RelB accumulation in the cytoplasm were detected in L428 cells. Transient transfection of TRAF3-expression vector enforced the expression of TRAF3 and blocked the p100 processing in L428 cells. The alternative NF-kB activity was partially decreased whereas the classical NF-kB activity remained intact. In addition, the increased TRAF3 expression did not affect the anti-apoptotic effects in L428 cells. Conclusions Not only the classical NF-κB activity but also the alternative NF-κB activity characterized by p100 processing and p52-RelB nuclear localization is constitutively activated in B cell-derived lymphoma cells. Lack of TRAF3 expression might be one of the reasons for the aberrant expression of alternative NF-κB activity. TRAF3 is indeed an important molecule regulating the activation of the alternative NF-kB activity but not the classical NF-kB activity in H-RS cells. Disclosures: No relevant conflicts of interest to declare.
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Milhollen, Michael, Usha Narayanan, Allison J. Berger, Michael Thomas, Tary Traore, Jie Yu, Julie Zhang, et al. "MLN4924, a Novel Small Molecule Inhibitor of Nedd8-Activating Enzyme, Demonstrates Potent Anti-Tumor Activity in Diffuse Large B-Cell Lymphoma." Blood 112, no. 11 (November 16, 2008): 606. http://dx.doi.org/10.1182/blood.v112.11.606.606.
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Abstract MLN4924 is a first-in-class, small molecule inhibitor of the Nedd8 Activating Enzyme (NAE) in Phase I clinical trials in hematological malignancies. Inhibition of NAE by MLN4924 leads to decreased neddylation and inhibition of cullin-dependent ubiquitin ligase (CDL) activity. CDLs are enzyme complexes which control the ubiquitination and degradation of proteins with important roles in cell cycle progression and cell survival. CDL-mediated degradation of pIkBa regulates NF-kB signaling by freeing cytoplasmic NF-kB transcription factors to translocate to the nucleus promoting cell proliferation and survival. In tumors dependent on the NF-kB pathway for growth and survival, we hypothesized that MLN4924 inhibition of CDL activity would prevent pIkBa degradation and inhibit NF-kB signaling. We utilized models of ABC-like Diffuse Large B-cell Lymphoma (ABC-like DLBCL, OCI-Ly10 and OCI-Ly3 cells) dependent on NF-kB signaling for survival and Germinal Center B-cell like DLBCL (GCB-like DLBCL, OCI-Ly19 and OCI-Ly7 cells) that are not dependent on NF-kB signaling for survival. In vitro, we show that NAE inhibition by MLN4924 in ABC-like DLBCL produces marked stabilization of pIkBa, inhibits p65 nuclear translocation and NF-KB gene transcription demonstrating an inhibition of NF-kB signaling. The inhibition of NF-KB signaling in Ly10 cells results in a G1 phenotype and an acute induction of apoptosis. In contrast, in GCB-like DLBCL we observed an elevation of multiple substrates of the CDLs, an accumulation of cells with increased DNA content (>4N) followed by a DNA damage response and induction of cell death. This mechanism of action in GCB-like DLBCL cells is observed in other tumor cell lines that are not dependent on NF-kB signaling for survival. In vivo administration of MLN4924 to mice bearing xenograft tumors of OCI-Ly10 and OCI-Ly19 resulted in a pharmacodynamic response of NAE pathway inhibition. In both models, a single dose of MLN4924 resulted in time and dose-dependent inhibition of total neddylated cullin levels and stabilization of CDL substrates including the CDL3Keap1 substrate, Nrf-2. Notably, in the OCI-Ly10 model, a single dose of MLN4924 resulted in a marked elevation of pIkBa levels, indicative of NF-kB pathway inhibition, and induction of apoptosis. In both OCI-Ly10 and OCI-Ly19 xenograft models, inhibition of the NAE pathway following repeated daily and intermittent dosing of MLN4924 translated into significant tumor growth inhibition. In the OCI-Ly10 model tumor regressions were observed showing this model to be particularly sensitive to MLN4924 treatment, reflecting the addiction of these tumors to NF-kB signaling. Additionally we demonstrate an inhibition of the NAE pathway and NF-KB signaling in a primary human tumor DLBCL xenograft model (PHTX-22L) resulting in tumor regressions following MLN4924 treatment. In summary, in tumors dependent on NF-kB signaling for growth and survival, MLN4924 inhibition of CDL activity provides a novel mechanism for targeted NF-kB pathway modulation and therapeutic intervention. In addition, these data demonstrate that MLN4924 is a novel agent that has broad activity in pre-clinical models of lymphoma.
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Kashyap, Trinayan, Boris Klebanov, Christian Argueta, Margaret S. Lee, Sharon Shacham, Michael Kauffman, Yosef Landesman, and William Senapedis. "Efficacy of Selinexor Is Dependent on IκB-α Expression and NF-Kb Deactivation in Multiple Myeloma Cells." Blood 128, no. 22 (December 2, 2016): 5660. http://dx.doi.org/10.1182/blood.v128.22.5660.5660.
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Abstract Background: Selinexor is a Selective Inhibitor of Nuclear Export (SINE) compound that binds to and inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) including p53, pRB, and IκB-α. Selinexor has therapeutic benefit both pre-clinically and clinically (NCT01607892, NCT02336815) in multiple myeloma (MM). It has been previously demonstrated that the extent of NF-κB transcriptional inhibition is one of the critical mechanisms contributing to the efficacy and/or resistance to selinexor in cells. However, the mechanism leading to NF-κB inhibition after selinexor treatment is not fully understood. We hypothesized that the level of the cellular inhibitor of NF-κB, IκB-α, and its compartmental localization play an important role in NF-κB transactivation and response to selinexor. In this study, we investigate the effect of selinexor treatment on MM cells with low expression of IκB-α and high NF-kB activity in order to understand the mechanism of NF-κB inhibition by selinexor. Methods: IM9 and RPMI-8226cells were treated with selinexor in the presence or absence of 20 ng/mL tumor necrosis factor α (TNFα; inflammatory mimic) and whole protein lysates were analyzed by immunoblotting. Cytotoxic effects of selinexor were evaluated using standard viability assays. IκB-α knockdown was performed using transfection of specific siRNA duplexes. NF-κB transcriptional activity was analyzed using an ELISA assay. Results: Selinexor induces nuclear localization of IκB-α in MM cells. RNAi of IκB-α in MM cells reduced the cytotoxic effects of selinexor by 10-fold. In addition, knockdown of IκB-α reduces the synergy of the selinexor plus proteasome inhibitor (bortezomib or carfilzomib) combination. This data suggests that in MM, IκB-α plays a major role in cellular sensitivity to selinexor potentially through NF-κB activity. Selinexor inhibited NF-kB transcriptional activity in IM9 and RPMI-8226 cells with IC50 of 1079 nM and 591 nM respectively. Although the difference in NF-κB activity IC50 is only 2-fold between the two MM cell lines (MTT IC50s are ~100 nM), IM9 cells have a 100-fold higher basal NF-kB activity when compared to RPMI-8226 cells. Under TNFα stimulation NF-kB activity was induced by 1.5- and 35-fold in IM9 and RPMI-8226, respectively. We observed that selinexor treatment caused a dose dependent inhibition of IκB kinase (IKK)-mediated phosphorylation of serine 32/36 on IκB-α and serine 536 on the NF-κB p65 subunit (RelA) upon TNFα stimulation in both cell lines. In RPMI-8226 cells, selinexor reduced TNFα-induced IκB-α phosphorylation in a dose dependent manner and protected IκB-α from degradation. In IM9 cells that have high basal NF-κB activity, TNFα did not induce NF-kB activity or cause IκB-α degradation. However, selinexor treatment inhibited NF-kB activity below its basal level (70% reduction) which resulted in dose dependent reduction in the level of IκB-α protein perhaps through inhibition of NF-κB transcriptional control of IκB-α mRNA expression. Conclusions: IκB-α plays a major role in the cellular cytotoxicity of selinexor in cancer cells. Multiple myeloma cells lose sensitivity to selinexor treatment upon IκB-α silencing, which in turn reduces the cytotoxicity of selinexor. TNFα stimulation induces the phosphorylation of NF-κB p65 subunit and IκB-α through increased IKK activity resulting in IκB-α degradation and NF-κB activation. In RPMI-8226 cells, selinexor treatment blocked TNFα-induced degradation of IκB-α. However, in IM9 cells TNFα alone did not have any significant effect on IκB-α which might be due to the high basal NF-kB activity. Interestingly, IκB-α is also a transcriptional target of NF-kB. In IM9 cells, selinexor treatment reduces NF-kB activity below the high basal level in a dose dependent manner resulting in near complete inhibition of NF-kB-controlled IκB-α mRNA transcription and a loss of the IκB-α protein. Ultimately, selinexor treatment inhibits cell viability and NF-kB transcriptional activity regardless of basal NF-κB activity in MM cells. Because of this IκB-α/NF-kB transcriptional mechanism, selinexor treatment can inhibit both chronic (unresponsive to TNFα) and acute (TNFα-simulated) inflammatory signaling which makes selinexor an applicable therapy to cancer cells with a variety of aberrant signaling pathways. Disclosures Kashyap: Karyopharm Therapeutics: Employment, Equity Ownership. Klebanov:Karyopharm Therapeutics: Employment, Equity Ownership. Argueta:Karyopharm Therapeutics: Employment, Equity Ownership. Lee:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Senapedis:Karyopharm Therapeutics: Employment, Equity Ownership.
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Kagoya, Yuki, Akihide Yoshimi, Shunya Arai, Keisuke Kataoka, Masahiro Nakagawa, Keiki Kumano, and Mineo Kurokawa. "NF-κB/TNF-α Positive Feedback Loop with Active Proteasome Machinery Supports Myeloid Leukemia Initiating Cell Capacity." Blood 120, no. 21 (November 16, 2012): 654. http://dx.doi.org/10.1182/blood.v120.21.654.654.
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Abstract Abstract 654 Acute myeloid leukemia (AML) is an aggressive hematologic malignancy arising from leukemia initiating cells (LIC), and is comprised of highly heterogeneous groups with different cytogenetic and molecular abnormalities, which makes it difficult to establish a broadly effective therapeutic strategy. Since constitutive NF-kB pathway activation has been reported in different types of AML cells, it is one of the promising candidates which are universally involved in the LIC phenotype. However, the mechanism of activation and its significance in leukemia progression have not been studied well. In this study, we explored NF-kB pathway activity and its role in LICs using various myeloid leukemia mouse models including MLL-ENL, MOZ-TIF2, and BCR-ABL/Nup98-HoxA9 leukemias. A number of NF-kB target genes showed elevated expression in LIC of each model: leukemic granulocyte-monocyte progenitors (L-GMP) in MLL-ENL or MOZ-TIF2 model and lineage- Sca-1+ fraction in BCR-ABL/Nup98-HoxA9 model, compared with normal hematopoietic stem cells (HSC) and GMP. Moreover, in immunofluorescence staining, each type of LIC displayed prominent nuclear translocation of NF-kB subunit p65. On the other hand, p65 was localized mainly in the cytoplasm in normal cells and, interestingly, non-LIC fraction in the bone marrow cells of the leukemia mice. To our surprise, LIC retained NF-kB activity even after serum-free culture, indicating that NF-kB pathway is prevalently activated in LIC in an autonomous fashion. To study the mechanism of activation, we analyzed gene expression profiles of LIC and normal HSC in murine and human AMLs and found that LIC showed distinctly elevated expression of TNF-a, one of the major activators of the NF-kB pathway. Consistent with these results, the culture media conditioned by LIC had higher TNF-a levels than those of normal cells. In all of the three types of leukemia mice, bone marrow extracellular fluid included higher TNF-a than that of control mice. Importantly, TNF-a blockage by the neutralizing antibody significantly attenuated p65 nuclear localization in LIC, and NF-kB inhibition by expression of a super repressor form of IkBa (IkB-SR) suppressed the TNF-a expression in LIC, indicating that LIC maintains its NF-kB activity by autocrine NF-kB/TNF-a positive feedback loop. Disruption of this loop by induction of IkB-SR or shRNA-mediated knockdown of TNF-a significantly reduced colony-forming abilities of leukemia cells and prolonged survival of leukemic mice in all the three models. In contrast, transduction of IkB-SR into normal HSC exerted no influence on their colony-forming ability. These results suggested that the NF-kB/TNF-a positive feedback loop plays a vital role for LIC propagation. We also addressed the mechanism of the difference in NF-kB activity between LIC and non-LIC. Notably, LIC had decreased protein levels of IkBa compared with non-LIC in spite of the same mRNA expression levels between them. In addition, basal 20S proteasome activity and the expression levels of proteasome subunit genes in LIC were higher than those in non-LIC, indicating that enhanced proteasomal degradation of IkBa could lead to selectively high NF-kB activity of LIC. The same propensity was seen in human AML CD34+CD38- cells versus CD34- cells in the analysis of microarray expression data. Proteasome inhibition by bortezomib diminished the differences of IkBa protein level. Moreover, its administration to leukemic mice selectively killed LIC fraction and prolonged survival in the in vivo transplantation model. Finally, forcible maintenance of NF-kB activity in LIC by shRNA-mediated knockdown of IkBa significantly enhanced its self-renewal activity as determined by surface marker profiles after in vitro culture. In the analysis of mice reconstituted with the IkBa-downregulated leukemic cells, bone marrow mononuclear cells had increased colony-forming cell ability and enhanced LIC frequency as determined by in vivo limiting dilution serial transplantation assay. These results indicated that the transition from LIC to non-LIC might be associated with the attenuation of NF-kB activity due to inefficient degradation of IkBa. In summary, these findings elucidate that NF-kB/TNF-a signaling in LIC, under support of the proteasome activity, has a critical role for both maintenance and propagation of LIC and provide a widely applicable approach for targeting LIC in myeloid leukemias. Disclosures: No relevant conflicts of interest to declare.
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Messa, Emanuela, Ilaria Defilippi, Antonella Roetto, Francesca Messa, Francesca Arruga, Sonia Carturan, Valentina Rosso, Enrico Bracco, Daniela Cilloni, and Giuseppe Saglio. "Deferasirox Is the Only Iron Chelator Acting as a Potent NF-KB Inhibitor in Myelodysplastic Syndromes." Blood 112, no. 11 (November 16, 2008): 2671. http://dx.doi.org/10.1182/blood.v112.11.2671.2671.
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Abstract Iron overload is a critical issue for low risk myelodisplastic syndrome (MDS) patients with a long tranfusional history and often requires chelation therapy. Iron chelation is as an independent prognostic factor for survival in MDS but can also improve haemoglobin level in some cases with different drugs and modalities. Recently a once daily oral chelator Deferasirox became available for the treatment of secondary hemosiderosis also in MDS patients and it has been described an haemoglobin improvement in a many patients within a few months of treatment. Moreover it was demonstrated that the transcriptional factor NF-kB is abnormally activated in MDS blast cells. Its pathway can be mediated by a broad variety of stimuli, sometimes dependent by reactive oxygen species generated by iron overload but it is activated even in the absence of iron overload. Aim of our study was to compare the effects of the 3 commercially available iron chelators on NF-kB activity in MDS and to identify a possible mechanism responsible for the observed reduced transfusion requirement during iron chelation therapy. We collected 40 PB samples from MDS patients: 18 RA, 14 RAEB, and 8 s-AML. Thirty of them presented hepatic iron overload (measured by SQUID biomagnetic liver susceptometry) and serum ferritin levels over 5000 ng/ml. Ten samples were collected before starting transfusion therapy. MNC cells were incubated with 50 microM Deferasirox for 3 hrs. K562 and HL60 cells were analyzed as controls and were incubated with Deferasirox 50 mM, Deferiprone 0,3 mM and Deferioxamine 0,3 mM for 18 hours and with the DL-Dithiothreitol (DTT) 100 mM for 18 hours as control. NF-kB activity was evaluated using both EMSA and ELISA methods. Apoptosis was evaluated by FACS for the detection of annexin V. Deferasirox incubation induced a significant decrease of NF-kB activity both in HL60 and K562 cells lines (EMSA method). We detected an increased activation of NF-kB as compared to healthy subjects in 6 RA, 12 RAEB, in all the s-AML PB samples and in cell lines. No significant difference was detected in NF-kB activity by comparing patients with or without iron overload (p=0,5). The percentage of samples presenting NF-kB activity increases during disease progression being higher in RAEB and s-AML as compared to RA (p=0,003). Among patients with increased NF-kB (n=14) the incubation with Deferasirox induced a significant reduction of NF-kB activity (p=0,0002). The degree of NF-kB inhibition was not significantly different according to the level of iron overload. Apoptosis was not significantly triggered by incubation with any of the different chelators. HL60 and K562 cells incubation with the Deferiprone and Deferioxamine failed to induce a significant reduction of NF-kB activity. NF-kB is abnormally activated in MDS patients and this activity is not strictly related to iron overload being present in patients with normal serum ferritin levels and in cell lines. Deferasirox is the only commercially available iron chelator acting as a potent NF-kB inhibitor and this effect is not shared by Deferioxamine, Deferiprone or DTT. This behaviour suggests that it is independent from ROS scavenging that is a common feature of all the mentioned drugs. Our in vitro observation could be an explanation for the early improvement of hemoglobin levels observed in some patients under deferasirox chelation that seems not related to a sharp decrease of iron overload.
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Timar, Botond, Amy Chadburn, Daniel Knowles, and Ethel Cesarman. "Activation of Classical and Alternative Nuclear Factor-kappaB (NF-kB) Pathways in Diffuse Large B-Cell Lymphomas." Blood 104, no. 11 (November 16, 2004): 29. http://dx.doi.org/10.1182/blood.v104.11.29.29.
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Abstract Activation of the NF-kB pathway is involved in many human neoplasms. In this study we examined the status of the NF-kB canonical (IkB, p50/p65) and non-canonical (p52, RelB) pathways in diffuse large B-cell lymphomas (DLBCL), which are a common and heterogeneous group of lymphoid malignancies. DLBCL have been divided into activated B-cell (ABC) like, and germinal center B-cell (GCB) like subgroups, which have been reported to have high and low NF-kB activity, respectively. However, the nature of the NF-kB complexes in this lymphoma entity has not been previously evaluated. Therefore we performed Western blotting, electrophoretic mobility shift assays (EMSA) and real time quantitative RT-PCRs on nuclear and cytoplasmic protein and total RNA extracts in 20 primary tumor samples and 9 different DLBCL cell lines. In the cell lines, presence of NF-kB proteins in nuclear extracts correlated with expression of NF-kB target genes (CCR7, IkBa, CCND2, BCL-2, IRF4) as determined by RT-PCR. Therefore, these could be assigned into GCB and ABC-like categories. In primary DLBCLs, EMSA showed NF-kB binding in all but one case. The same cases (19/20) had high p52 in the nucleus indicating activation of the alternative pathway. TNF family ligand BAFF was also found to be expressed in all primary samples and most cell lines. The classical pathway, as determined by nuclear p50, was also present in these cases. Levels of p65 and RelB expression were variable, but did not correlate with the mRNA expression of NF-kB target genes. In conclusion, while DLBCL cell lines may be divided into two distinct categories, the primary samples represented a spectrum of NF-kB target gene activity, while levels of NF-kB expression were high. BAFF expression and activation of the alternative pathway may be important in the pathogenesis of DLBCL.
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Pham, Lan V., Archito Tamayo, Hai-Jun Zhou, Yen-Chiu Lin Lee, Lingchen Fu, Carlos E. Bueso-Ramos, Elias Drakos, L. Jeffrey Medeiros, and Richard J. Ford. "Networking Modules of Canonical and Alternative NFkB Signaling in Growth and Survival Regulations of Large B Cell Lymphomas." Blood 112, no. 11 (November 16, 2008): 3776. http://dx.doi.org/10.1182/blood.v112.11.3776.3776.
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Abstract Constitutive NF-kB activation is considered a “hallmark” in B-cell malignancies, especially in large B-cell lymphoma (LBCL), a common but heterogenous Non-Hodgkin’s lymphoma type. However, the mechanism(s) of activation and interactions of NF-kB components in the nuclear compartment of B-cell lymphomas are still poorly defined. Our findings demonstrate that both the canonical and the alternative NF-kB pathways are constitutively activated in both LBCL cell lines and primary lymphoma cells. NF-kB DNA binding ELISA analysis showed that the canonical NF-kB components p65 and c-rel as well as the non-canonical NF-kB components p52 and relB are constitutively activated in 14 LBCL cell lines and in 14 primary lymphoma patients’ cells (both ABC-and GCB-like subtypes). Based on the ELISA data, we found that the p65/c-rel ratio is relatively higher in ABC-like LBCL subtype when compared to GCB-like LBCL subtype, but the alternative NF-kB members’ p52 and rel-B are differentially activated in both GCB- and ABC-like LBCL subtypes. Micro-tissue array (MTA) studies confirmed that both NF-kB pathways are constitutively active in LBCL biopsy-cores. The components of both NF-kB pathways were also found to merge and form functional “hybrid” NF-kB complexes in the nuclear compartment. Besides the common hetero-dimeric complexes occur between p50, p65, and c-rel of the canonical NF-kB pathway and between p52 and relB of the alternative NF-kB pathway, components of both pathways also merged to form “hybrid” dimeric complexes, such as p52-relB, p52-c-rel, relB-p65, and relB-c-rel in LBCL cells. Interestingly, p65 and c-rel binds to relB and p52, respectively, with higher affinity in GCB- than in ABC-like LBCL subtype. Our data also showed that activation of both NF-kB pathways and their cross-interaction are not restricted to only neoplastic B cells but that they are also activated and utilized in highly proliferative activated normal B-lymphocyte. Specific siRNA that target individual NF-kB members p65, c-rel, and rel-B have inhibitory activity in lymphoma cell growth, but similar p52 siRNAs do not have inhibitory activity. These studies indicated that in both LBCL cell lines and patient samples (GCB-and ABC-like subtypes), both the canonical and the alternative NF-kB pathways are not only constitutively activated but also interact to form multimeric NF-kB complexes in the nuclear compartment, giving rise to an NF-kB signaling module that not only controls lymphoma cell growth and survival but likely contributes to the heterogeneity of the disease processes. These findings reveal additional aspects of molecular heterogeneity and complexity in NF-kB signaling mechanisms and interacting transcriptional modules, contributing to the pathophysiology of LBCL. These results also highlight the critical importance of multiple constitutively active NF-kB signaling pathways in LBCL and may also contribute to therapeutic refractoriness, particularly in relapsed/refractory LBCLs.
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Tiedemann, Rodger E., Jonathan J. Keats, Jessica Schmidt, Chang-Xin Shi, Yuan X. Zhu, Xinliang Mao, Aaron D. Schimmer, and Keith Stewart. "Identification of a Potent Natural Triterpenoid Inhibitor of Proteosome Chymotrypsin-Like Activity and NF-κB with Specific Anti-Myeloma Activity in Vitro and in Vivo." Blood 112, no. 11 (November 16, 2008): 3679. http://dx.doi.org/10.1182/blood.v112.11.3679.3679.
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Abstract As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that inhibit signaling pathways driving cyclin D2 promoter transactivation, assaying more than 4,000 compounds. The top-ranked compound from these studies was a natural triterpenoid, pristimerin. Pristimerin markedly suppressed cyclin D2 promoter activity (>90%) in 3T3 fibroblast cells and inhibited cyclin D1, D2 and D3 protein expression in myeloma tumor cells. Strikingly, the early (4 hour) transcriptional response of myeloma cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors (P<10−9) (Connectivity Map Build 2, www.broad.mit.edu/cmap), with rapid induction of heat shock proteins (HSP70 >90-fold), activating transcription factor (ATF) 3 and CHOP. Enzymatic assays performed with purified 20S proteosome, or with total cellular extract, confirm that pristimerin rapidly and specifically inhibits chymotrypsin-like 20S proteosome activity at low concentration (<100nM), causing sustained inhibition lasting >6 hours. Consistent with inhibition of proteosome function, pristimerin causes rapid and sustained accumulation of high molecular weight poly-ubiquitinated protein in myeloma cell lines. Notably, related cytotoxic triterpenoid drugs, such as the methyl ester of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me, RTA 402) or betulinic acid or the ginsenosides - all of which show promising anti-cancer activities and are currently in clinical trials for advanced lymphoma, leukemia or solid malignancies - commonly inhibit NF-kB activation via direct inhibition of IKKα or IKKβ. In contrast drugs that function as proteosome inhibitors also commonly suppress NF-kB function instead by impairing degradation of ubiquitinated IkB. Immunoblotting for phosphorylated IkB confirms that pristimerin, like other triterpenoids, acts upstream of IkB to inhibit its phosphorylation, although pristimerin simultaneously inhibits proteosome activity with marked potency to diminish the clearance of ubiquitinated IkB. As a result of this two-fold stabilization of IkB, pristimerin causes overt and specific suppression of NF-kB mediated transcription, measured by a panel of transcriptional reporters with synthetic promoters containing 5x repeats of generic binding sites for NF-kB, AP-1, CREB or TCF4. Importantly, specific suppression of constitutive NF-kB transcriptional activity was pronounced in myeloma cells with inherent NF-kB pathway activation resulting from bi-allelic deletion of the TRAF3 tumor suppressor. Constitutive activation of the NF-kB pathway occurs in a significant proportion of primary myeloma tumors, most commonly via inactivation of TRAF3. Selective silencing of NF-kB driven transcription in myeloma cells may mediate the potent suppression of cyclin D proteins induced by this compound. Significantly, multiple myeloma cells are exquisitely sensitive to both proteosome inhibition or NFkB pathway inhibition. Consistent with these twin vulnerabilities, pristimerin is potently and selectively lethal to primary myeloma cells from patients (IC50<100nM) grown in mixed lineage culture and inhibits the growth of xenografted human plasmacytoma tumors grown in mice, providing a strong rationale for pharmaceutical development of triterpenoid dual-function proteosome-plus-NF-kB pathway inhibitors as therapeutics for multiple myeloma and related human malignancies.
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Guo, Feng, Zi-Xing Chen, Aining Sun, Peng Zhou, and Wenjuan Wang. "Differential Effects of TRAFs in the Activation of NF-KappB in Hodgkin’s Lymphoma Cells." Blood 112, no. 11 (November 16, 2008): 1458. http://dx.doi.org/10.1182/blood.v112.11.1458.1458.
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Abstract CD30, a member of the TNF receptor (TNFR) superfamily, is a lymphocyte-specific receptor that is originally recognized as a surface molecule overexpressed on Hodgkin’s lymphoma cells. Engagement of CD30 with its ligand CD30L activates the nuclear factor kB (NF-kB), which are mediated by interactions with TNFR-associated factor (TRAFs), and supports proliferation of Hodgkin’s cells. However, the role of individual TRAFs in the CD30 signaling pathway in Hodgkin’s cells has not been fully addressed yet. In this study, we found that except TRAF3, all other TRAFs were expressed consistently in B cell-derived Hodgkin’s cell lines (L428 and KM-H2) either in mRNA or protein level. In L428 but not KM-H2 cells, the classical (p50-RelA) and alternative (p52-RelB) NF-kB activity were constitutively activated measured by Western Blotting and EMSA. To better understand the CD30 signaling properties in Hodgkin’s lymphoma cells, we silenced the expression of TRAFs individually by means of RNAi. Successful downregulation of TRAF1 protein expression led to the apoptosis of L428 cells, which was companied by the reduction of both classical and alternative NF-kB activity. Furthermore, the expression of targeting genes of NF-kB, such as A20, c-Flip, ICAM-1, and Cyclin D1 was also decreased. Using siRNA targeting TRAF2 expression, the classical NF-kB activity was reduced while the alternative NF-kB activity moderately induced especially upon CD30L treatment, which was companied by the induction of p100 processing and RelB nuclear localization. The survival rate was decreased when TRAF2 was knockdown. TRAF5 knockdown manifested similar results as that of TRAF2. These observations were only took place in L428 cells but not KM-H2 cells. In addition, the phosphorylation of the extracellular signal-regulated kinases (ERK) 1 and 2, and subsequent activation of JunB in Hodgkin’s cells were staying unchanged in individual TRAFs knockdown experiment. Taken together, the study here further investigates the features of CD30-TRAFs-NF-kB signaling pathway in Hodgkin’s lymphoma cells. At least TRAF1, 2, and 5 are involved in CD30 signaling pathway and protecting Hodgkin’s lymphoma cells from undergoing apoptosis. The interaction between TRAFs is necessary to facilitate the signaling and proper activation of classical NF-kB, which is responsible for the antiapoptotic effect of Hodgkin’s cells. Among TRAF1, 2, and 5, the redundant function is unlikely. Furthermore, the sequential activation of ERK and JunB is unlikely dependent on TRAFs-NF-kB pathway in Hodgkin’s lymphoma cells. Thus, these data reveal that the classical NF-kB activity downstream of CD30 signaling is TRAFs-dependent in Hodgkin’s lymphoma cells, which is regulated by the relative level of individual TRAF in a cell-specific manner.
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Yang, Jun, Francis R. Leblanc, Shubha Dighe, Susan B. Nyland, David J. Feith, and Thomas P. Loughran. "Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Mediates and Sustains NF-κb Constitutive Activation in LGL Leukemia Cells." Blood 128, no. 22 (December 2, 2016): 2762. http://dx.doi.org/10.1182/blood.v128.22.2762.2762.
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Abstract Large granular lymphocyte (LGL) leukemia results from clonal expansion of CD3+ cytotoxic T-lymphocytes (CTL) or CD3-natural killer (NK) cells. Chronic antigen stimulation promotes long-term survival of LGL leukemia cells through constitutive activation of multiple survival pathways, leading to global dysregulation of apoptosis. Clinical manifestations of LGL leukemia include neutropenia, anemia and rheumatoid arthritis. Treatment for LGL leukemia patients relies on immunosuppressives such as methotrexate and is not curative. No standard therapy has been established. We reported that nuclear factor kappa B (NF-kB) is central to the leukemic LGL survival network, but the mechanisms of constitutive NF-kB activation in LGL leukemia are undefined. TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis and activates NF-kB via binding to TRAIL receptor (TR) 1, 2 and decoy receptor 2 (DcR2). DcR2 is uniquely unable to transduce downstream death signals yet retains NF-kB transduction activity. The mechanisms of TRAIL expression and regulation in LGL leukemia are unknown. Thus the current study investigates these mechanisms and their potential therapeutic applications, with the use of NF-kB inhibitors ixazomib and bortezomib, in LGL leukemia. Methods: LGL leukemia cell lines TL1 (T-LGL) and NKL (NK-LGL), peripheral blood mononuclear cells (PBMC) from LGL leukemia patients, and PBMC from normal donors were studied. NF-kB DNA binding activity was determined by EMSA. Results were confirmed using probe-based NF-kB (p50/65) transcription factor assay and immunocytochemistry (ICC). Serum TRAIL levels were detected by ELISA. Cellular TRAIL expression was determined by real-time PCR, western blot and ICC. DcR2 and Mcl-1 siRNA knock-down was performed with electroporation. Flow cytometry was used to detect TR 1-3 and DcR2 expression and apoptosis. Results: The average serum levels of TRAIL in LGL leukemia patients were nearly 4-fold higher than normal (NL) control values (p ≤ 0.0001). Data from RT-PCR (p ≤ 0.04), western blot and ICC revealed that LGL leukemia cells were the major source of TRAIL overexpression. Identical expression levels of TR1, 2 and 3 were observed in PBMC from LGL leukemia patients and from NL controls. Like normal PBMC, LGL leukemia cells were resistant to TRAIL-induced apoptosis. In contrast, the expression frequency of DcR2 was at least 4-fold greater in LGL leukemia PBMC compared to NL control, and it correlated to the percentage of circulating LGL leukemia cells. We found that TRAIL activates NF-kB and NF-kB downstream target genes, including TRAIL and McL-1, in LGL leukemia samples. To confirm that TRAIL is responsible for constitutive NF-kB activation in LGL leukemia, T-LGL leukemia PBMC were treated with pooled sera from 3 each of either NL controls or T-LGL leukemia patients. Leukemia sera increased NF-kB activity on EMSA, and this effect was completely blocked by TRAIL neutralizing antibody. DcR2 siRNA knockdown specifically decreased RelA and NF-kB1 (p105/p50) levels in TL1 and NKL cells. Mcl-1 siRNA mediated increased apoptosis in the same cell lines. Likewise, ixazomib and bortezomib facilitated leukemia-selective apoptosis in LGL leukemia cell lines and in patient PBMC, via inhibition of NF-kB activity and of downstream targets (ixazomib, p≤0.0001; bortezomib, p≤0.03). Additionally, caspase-3 and PARP cleavage were observed in LGL leukemia cells treated with ixazomib or bortezomib. Serum TRAIL levels in LGL leukemia patients were significantly lower in methotrexate responders versus non-responders, corresponding with reduced NF-kB DNA binding activity and increased absolute neutrophil counts, indicative of treatment response. Conclusion: These data indicate that expression of DcR2 and constitutive activation of NF-kB are responsible for TRAIL resistance in leukemic LGLs. TRAIL triggers prolonged NF-kB activation via interaction with DcR2, and activated NF-kB in turn promotes further TRAIL production in leukemic LGLs, creating a TRAIL autocrine regulatory loop. Inhibition of NF-kB activity with ixazomib and bortezomib interrupts this loop, impairs expression of Mcl-1 and induces apoptosis of leukemia cells. Our preclinical findings provide a solid framework for clinical evaluations of ixazomib and bortezomib in the treatment of LGL leukemia. Disclosures No relevant conflicts of interest to declare.
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Hewamana, Saman, Clare Rowntree, Thet Thet Lin, Chris Pepper, Paul Brennan, and Chris Fegan. "Constitutive Nuclear p65 NF-kB Expression Predicts for Spontaneous Apoptosis and In Vitro Sensitivity to Fludarabine in CLL Cells." Blood 108, no. 11 (November 16, 2006): 4974. http://dx.doi.org/10.1182/blood.v108.11.4974.4974.
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Abstract B-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B-cells in the blood, secondary lymphoid tissues, and bone marrow. The leukemia cells primarily are arrested in the G0/G1 phase of the cell cycle and appear resistant to programmed cell death. Despite their apparent longevity in vivo, CLL cells typically undergo spontaneous apoptosis in vitro although there is considerable heterogeneity in the timing and extent of this process. In this study we investigated the degree of this apoptosis in vitro, and its association with constitutive nuclear p65 NF-kB expression (n = 25). Apoptosis was measured by annexin V / propidium iodide labeling using flow cytometry and nuclear NF-kB expression was evaluated using electrophoretic mobility shift assay and quantified using a p65 ELISA. In contrast to previous reports, we found constitutive NF-kB activity varied in our patient cohort with a trend towards higher expression in samples with unmutated VH genes, high CD38 expression and high ZAP-70 expression. Importantly, we demonstrated an inverse correlation between the percentage of Annexin V-positive cells and nuclear p65 NF-kB expression (r2 = − 0.21; P = 0.02) suggesting that high levels of p65 NF-kB are cytoprotective in CLL cells. We then investigated the relationship between p65 NF-kB and in vitro response to fludarabine. Sensitivity to fludarabine was evaluated using LD50 values calculated from sigmoidal dose-response curves. These LD50 values positively correlated with nuclear p65 NF-kB activity (r2 = 0.25; P = 0.006) indicating that in vitro drug resistance to fludarabine may be mediated, at least in part, through the transcriptional effects of NF-kB. Taken together, our data indicate that NF-kB is a key regulator of both spontaneous and drug induced apoptosis in CLL cells and is therefore a promising therapeutic target for the treatment of this incurable condition.
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Cilloni, Daniela, Francesca Messa, Valentina Rosso, Sonia Carturan, Francesca Arruga, Ilaria Defilippi, Renata Catalano, et al. "NPM1 Mutations Are Responsible for Better Response to Induction Therapy in AML Patients through the Inactivation of NF-kB." Blood 106, no. 11 (November 16, 2005): 105. http://dx.doi.org/10.1182/blood.v106.11.105.105.
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Abstract Mutations in NPM1 exon 12 and the resulting shift of NPM1 into the cytoplasm are the most specific and frequent events in acute myeloid leukaemia patients with normal karyotype. Cytoplasmatic NPM is associated with responsiveness to induction chemotherapy, although its role in predicting outcome after remission remains to be defined. The aim of the study was to identify the molecular mechanisms responsible for chemosensitivity in leukemic cells carrying the mutation of NPM1. NF-kB is a transcription factor involved in many intracellular pathways including apoptosis. NF-kB is a heterodimer of p50 and p65 subunits sequestered in the cytoplasm in its inactive form through interaction with inhibitory IKB proteins and activated in the nucleous after degradation of IKB. The activation of NF-kB is responsible for chemoresistance to different drugs including anthracyclines. Based on this assumption, we analyzed the possible cytoplasmatic interaction between the mutated form of NPM1 (NPM+) and NF-kB. The NF-kB DNA binding activity was analyzed in 20 BM samples collected from AML patients carrying the NPM1 mutations and 30 NPM1 wild type samples (NPM1−) using an ELISA method. Immunofluorescence analysis using NPM1 and p65 antibodies was performed to identify the localization of both proteins. Western blot for p65 and NPM1 was used to confirm the protein amount and localization. Finally, co-immunoprecipitation assay was perform to study the interaction of the two proteins. We found a significant lower DNA binding activity in NPM1 mutated cells when compared to the wild type NPM cells (p=0,001). Immunofluorescence analysis confirmed the cytoplasmatic localization of NPM protein in mutated samples and the nuclear localization in wild type samples. In addition, immunofluoresence analysis performed with a monoclonal antibody against p65 subunit shows different pattern of NF-kB localization in NPM1+ when compared to NPM1−. In particular, in NPM1+ cells NF-kB is mainly localized in the cytoplasm in the inactive form and in NPM− cells is mainly nuclear localized. These data were confirmed by Western blot carried out with the same monoclonal antibody against p65 in the nuclear and cytosolic extracts. The interaction of NPM1 and NF-kB was further investigated and demonstrated by co-immunoprecipitation studies. In conclusion, we demonstrated that p65 and NPM1 interact with each other within the cytoplasm and this interaction results in the sequestration of NF-kB within the cytoplasm. The cytosolic localization of the inactive form of NF-kB explains the reduced NF-kB DNA binding activity observed in NPM1+ patients. These data may provide a possible explanation for the chemosensitivity observed in NPM+ patients. Furthermore, since NF-kB is involved in the transcription of many genes which regulate proliferation and differentiation processes, the disruption of NF-kB function may represent one of the mechanism of leukemogenesis induced by NPM1 mutated proteins.
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Islam, Abul, Ken-ichiro Otsuyama, Jakia Amin, Saeid Abroun, Karim Shamsasenjan, Mohd S. Iqbal, and Michio M. Kawano. "SDF-1 Is Responsible for the Constitutively High NF-kB Activity in Human Myeloma Cells." Blood 110, no. 11 (November 16, 2007): 4737. http://dx.doi.org/10.1182/blood.v110.11.4737.4737.
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Abstract The chemokine, stromal cell-derived factor 1 (SDF-1; CXCL12) and its receptor, CXCR4 are considered to be essentially required for plasma cell homing to the bone marrow (BM). It is well known that plasma cells in the BM (long-lived plasma cells) survive for a long time and have the constitutively high NF-kB activity. Since human myeloma cells are considered to be derived from these committed long-lived plasma cells, we investigated the role of SDF-1 on the survival of primary myeloma cells from myeloma patients and the possible relationship with NF-kB activity. First, we confirmed that all primary myeloma cells expressed CXCR4 but not CCR9 or CCR10 receptors on their surface and the levels of CXCR4 expression apparently correlated with maturity of BM plasma cells; mature myeloma cells (MPC-1+) as well as polyclonal plasma cells expressed higher levels of CXCR4 than those on immature myeloma cells (MPC-1-). The production of SDF-1 was found strongly in BM stromal cells but not in primary myeloma cells as well as myeloma cell lines. On the other hand, high DNA binding activity of NF-kB was constitutively detected in primary myeloma cells as well as myeloma cell lines, and these NF-kB activities significantly correlated with the expression levels of CD54 on their surface, for CD54 gene is one of the strict NF-kB target genes. Based on the expression levels of CD54 protein, interestingly, primary myeloma cells showed weaker NF-kB activities than those in monoclonal plasma cells from MGUS and polyclonal plasma cells from polyclonal gammopathy. Plasma concentrations of SDF-1 were also significantly correlated to the expression levels of CD54 on primary myeloma cells significantly (P<0.01). Furthermore, it was confirmed that addition of SDF-1 significantly increased the expression levels of CD54 in the in vitro culture of primary myeloma cells. Therefore, these results indicate that SDF-1 is responsible for high expression levels of CD54 and possibly the constitutively high NF-kB activity in primary myeloma cells.
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Hillman, K., J. Qian, J. N. Siegel, G. Roderiquez, R. Blackburn, J. Manischewitz, M. Norcross, and H. Golding. "Reduced susceptibility to HIV-1 infection of ethyl-methanesulfonate-treated CEM subclones correlates with a blockade in their protein kinase C signaling pathway." Journal of Immunology 148, no. 12 (June 15, 1992): 3991–98. http://dx.doi.org/10.4049/jimmunol.148.12.3991.
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Abstract We have described the isolation of chemically induced CEM subclones that express CD4 receptors and bind soluble gp120, yet show a markedly reduced susceptibility to infection with HIV-1. Two subclones were found to have an abnormal response to the protein kinase C (PKC) activator PMA. PMA treatment induced CD3 and CD25 (IL-2R) receptors on the parental line and on other ethyl-methanesulfonate-derived subclones, but not on these two mutants. Direct assays of PKC activity were conducted. Total cellular PKC enzymatic activity was found to be normal in these subclones. PMA-induced CD4 down-modulation occurred normally. In addition, activation of c-raf kinase was normal. Since HIV-1 long terminal repeat contains two functional nuclear factor kB (NF-kB) regulatory elements, we studied the ability of PMA to induce NF-kB binding activity by different assays. Chloramphenicol acetyl transferase (CAT) assays using the HIV-1 (-139)long terminal repeat-CAT construct showed no PMA induction of CAT activity in these subclones (unlike the parental line and other subclones). Okadaic acid, an inhibitor of phosphatases 1 and 2A, did not overcome the defect in these subclones. Gel retardation assays, using a 32P-probe containing the HIV-1 NF-kB probe and nuclear extracts from PMA-treated cells, showed significantly reduced induction of nuclear NF-kB binding proteins in these two subclones compared with wild type CEM and a control subclone. Deoxycholate treatment of cytoplasmic extracts from these subclones released much reduced NF-kB binding proteins from their cytoplasmic pools. Thus, reduced levels of PKC-induced nuclear NF-kB activity in two T cell subclones did not affect their normal cell growth, but correlated with a pronounced reduction in their susceptibility to HIV-1 infection.
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Dickson, Kathleen M., Asha L. Bhakar, and Philip A. Barker. "TRAF6-dependent NF-kB transcriptional activity during mouse development." Developmental Dynamics 231, no. 1 (2004): 122–27. http://dx.doi.org/10.1002/dvdy.20110.
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Pasqualucci, Laura, Mara Compagno, Wei Keat Lim, Adina Grunn, Subhadra V. Nandula, Marta Scandurra, Francesco Bertoni, et al. "Mutations in Multiple Genes Cause Deregulation of the NFkB Pathway in Diffuse Large B-Cell Lymphoma." Blood 112, no. 11 (November 16, 2008): 801. http://dx.doi.org/10.1182/blood.v112.11.801.801.
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Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease comprising multiple biologically and clinically distinct subgroups, including germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Gene expression profile studies have shown that a key feature of its most aggressive subtype, ABC-DLBCL, is the constitutive activation of the NF-kB transcription complex. However, except for a small fraction of cases (Lenz et al., Science 2008), it remains unclear whether NF-kB activation in these tumors reflects an intrinsic program of the cell of origin or represents a primary pathogenetic event. To address this question, we first characterized 165 DLBCL samples (18 cell lines and 147 primary biopsies, including 26 ABC, 28 GCB, 10 unclassified and 83 not profiled) for the presence of active, nuclear NF-kB complexes by using immunohistochemical/immunofluorescence staining of NFKB1 p105/p50 (as a readout for the canonical pathway) and NFKB2 p100/p52 (as a readout for the non-canonical pathway). Nuclear localization of NF-kB, indicative of constitutive activity, was observed in 14/26 (54%) ABC-DLBCL and 8/28 (28%) GCB-DLBCL primary biopsies, as well as in 3/10 (30%) unclassified and 48/83 (58%) non-profiled cases, and correlated with significant enrichment in expression of NF-kB target genes, as assessed by gene set enrichment analysis (GSEA) of transcriptionally profiled cases. In addition, the more sensitive GSEA approach detected a gene expression signature of NF-kB activity in >90% ABC-DLBCLs and 53% GCB-DLBCLs, indicating that constitutive activation of this key signaling pathway is a common feature of ABC-DLBCL but can also be observed in a smaller fraction of GCB-DLBCL. To investigate whether NF-kB activity represents a primary pathogenetic event, we then screened for mutations the complete coding sequences of 31 genes encoding for NF-kB pathway components in a panel of 14 ABC-DLBCLs, which was expanded to 48 samples (12 ABC, 26 GCB and 10 not profiled) for validation of the mutated genes. The results showed that >50% of ABC-DLBCL (n=15/26) and a smaller fraction of GCB-DLBCL (n=8/26, 31%) carry somatic mutations in multiple genes, including negative (TNFAIP3/A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7/TAK1 and TNFRSF11A/RANK) regulators of NF-kB. Of these, the A20 gene, which encodes for a ubiquitin-editing enzyme involved in termination of NF-kB responses, is the most commonly affected, with ~27% ABC-DLBCLs and 25% (8/32) immunohistochemically classified non-GC DLBCL displaying biallelic A20 inactivation by somatic mutations and/or deletions. Sequence changes include premature nonsense mutations, frameshift deletions/insertions and splice site mutations, leading to severely truncated proteins that lack functionally relevant domains and have thus lost their enzymatic activity. In virtually all mutated cases, FISH analysis revealed loss of the second allele, while 4 additional samples showed biallelic deletion of the gene. Thus, A20 is inactivated by a classic “two-hit” mechanism, suggesting a tumor suppressor role. Less frequently, missense mutations of CARD11 (10%) and TRAF2 (4%) produce molecules with significantly enhanced ability to activate NF-kB in transient transfection/reporter gene assays. Our results demonstrate that NF-kB activation in DLBCL is caused by genetic lesions affecting multiple genes, whose loss or activation may promote lymphomagenesis by leading to abnormally prolonged NF-kB responses. These findings provide the rationale and the assays for the identification of patients amenable to NF-kB targeted therapeutic intervention.
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Maeda, Yasuhiro, Terufumi Yamaguchi, Yasuki Hijikata, Yasuyoshi Morita, Chikara Hirase, Syunsuke Takai, Jun-ichi Miyatake, Mitsuhiro Matsuda, and Akihisa Kanamaru. "Dichotomy of All-Trans Retinoic Acid Inducing Signals for Adult T-Cell Leukemia." Blood 106, no. 11 (November 16, 2005): 4803. http://dx.doi.org/10.1182/blood.v106.11.4803.4803.
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Abstract We previously reported that all-trans retinoic acid (ATRA) inhibits growth in HTLV-1-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. However, the mechanism of this inhibition is not clear. In the present study, we observed that NF-κB transcriptional activity as well as cell growth decreased significantly in HTLV-1-positive T-cell lines in the presence of ATRA. Furthermore, we observed that ATRA reduced HTLV-1 proviral DNA, HTLV-1 genes (gag, tax or pol mRNA) using the real time quantitative polymerase chain reaction. SIL-2R was reduced by ATRA in both protein level (culture supernantant) and mRNA level in HTLV-1-positive T-cell lines. Interestingly, ATRA significantly inhibited RT activity similar to azidothimidine (AZT) in HTLV-1-positive T-cell lines. Moreover, AZT was inhibitory of proviral DNA but not NF-kB transcriptional activity and sIL-2R on HTLV-1, however ATRA was inhibitory of NF-kB, proviral DNA and sIL-2R on HTLV-1. These results suggested that the decrease of sIL-2R induced by ATRA may be caused by the actions of a NF-kB inhibitor acting on the NF-kB/sIL-2R signal pathway. These results suggested that ATRA could have two roles, as a NF-kB inhibitor and as a RT inhibitor.
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Togano, Tomiteru, Makoto Nakashima, Mariko Watanabe, Kazuo Umezawa, Toshiki Watanabe, Masaaki Higashihara, and Ryouichi Horie. "Inhibition of NF-κB Activation Augments the Effect of 5-Azacytidine On Acute Myeloid Leukemia Cell Line." Blood 120, no. 21 (November 16, 2012): 1369. http://dx.doi.org/10.1182/blood.v120.21.1369.1369.
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Abstract Abstract 1369 Dehydroxymethylepoxyquinomycin (DHMEQ) is a unique NF-kB inhibitor that is a 5-dehydroxymethyl derivative of novel compound epoxyquinomicin C and is currently under development for clinical use. We have shown that DHMEQ specifically binds to NF-kB having transactivation domain and inhibits NF-kB activation at the level of the nuclear translocation and the DNA binding. Constitutive activation of NF-kB has turned out to be a hallmark of various types of cancers originated from hematopoietic and other organs. We showed the effectiveness of DHMEQ in hematological malignancies with constitutive activation of NF-kB including multiple myeloma, chronic lymphocytic leukemia, adult T-cell leukemia, pleural effusion lymphoma and Hodgkin lmphoma. However, significance of NF-kB as a molecular target in acute myeloid leukemia (AML) is not well understood. Recently 5-azacytidine is widely used for the treatment of myelodysplastic syndrome and other cancers. 5-Azacytidine is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. Therefore it is possible that 5-azacytidine induces molecules required for NF-kB activation. However, the effect of 5-azacytidine on NF-kB induction, which may antagonize the anti-cancer effect is poorly understood. In this study we examined the constitutive NF-kB activation and the effectiveness of DHMEQ on AML cell lines, HL-60, ML-2, HEL, K562, CMK and Meg0-1. We also examined the induction of NF-kB by 5-azacytidine and evaluated the combined treatment of AML cell lines by 5-azacytidine and DHMEQ. AML cell lines except for K562 showed constitutive activation of NF-κB consisting of RelA and p50 subcomponent. The NF-κB binding activity observed in AML cell lines were relatively modest compared to that observed in Jurkat cells treated with tumor necrosis factor (TNF). Treatment of AML cell lines with constitutive activation of NF-κB by DHMEQ inhibited NF-κB and significantly reduced their viability. Treatment with 5-azacytidine also significantly reduced the viability of AML cell lines, however K562 showed resistance against the treatment. Addition of DHMEQ enhanced the effect of 5-azacytidine on cell viability of HL-60 and CMK cells. The treatment with 5-azacytidine enhanced NF-kB activation although the induction was modest and DHMEQ abolished both constitutive and induced NF-kB activation in HL-60 and CMK cells. The results indicate that NF-κB is a molecular target of AML cells and DHMEQ is a potential compound for the treatment of AML. Although the NF-κB activity is relatively low compared to that in lymphoid malignancies, the constitutive activation of NF-κB in AML cell lines appears to play a significant role for their survival. The results also indicate that 5-azacytidine can reduce viability of AML cells and combined use of an NF-κB inhibitor augments the effect of 5-azacytidine by inhibiting constitutive and induced NF-κB. Since NF-κB cooperates with various transcription factors and supports the survival of cells, interruption of the crosstalk between NF-kB and molecules induced by 5-azacytidine might also be responsible for the combined effect of DHMEQ and 5-azacytidine. Disclosures: No relevant conflicts of interest to declare.
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Stephenson, Diane, Tinggui Yin, E. Barry Smalstig, Mei Ann Hsu, Jill Panetta, Sheila Little, and James Clemens. "Transcription Factor Nuclear Factor-Kappa B is Activated in Neurons after Focal Cerebral Ischemia." Journal of Cerebral Blood Flow & Metabolism 20, no. 3 (March 2000): 592–603. http://dx.doi.org/10.1097/00004647-200003000-00017.
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Nuclear factor-kappa B (NF-kB) is a multisubunit transcription factor that when activated induces the expression of genes encoding acute-phase proteins, cell adhesion molecules, cell surface receptors, and cytokines. NF-kB is composed of a variety of protein subunits of which p50-and p65-kDa (RelA) are the most widely studied. Under resting conditions, these subunits reside in the cytoplasm as an inactive complex bound by inhibitor proteins, IkBα and IkBβ. On activation, IkB is phosphorylated by IkB kinase and ubiquitinated and degraded by the proteasome; simultaneously, the active heterodimer translocates to the nucleus where it can initiate gene transcription. In the periphery, NF-kB is involved in inflammation through stimulation of the production of inflammatory mediators. The role of NF-kB in the brain is unclear. In vitro, NF-kB activation can be either protective or deleterious. The role of NF-kB in ischemic neuronal cell death in vivo was investigated. Adult male rats were subjected to 2 hours of focal ischemia induced by middle cerebral artery occlusion (MCAO). At 2, 6, and 12 hours after reperfusion, the expression and transactivation of NF-kB in ischemic versus nonischemic cortex and striatum were determined by immunocytochemistry and by electrophoretic mobility gel-shift analysis. At all time points studied, p50 and p65 immunoreactivity was found exclusively in the nuclei of cortical and striatal neurons in the ischemic hemisphere. The contralateral nonischemic hemisphere showed no evidence of nuclear NF-kB immunoreactivity. Double immunofluorescence confirmed expression of p50 in nuclei of neurons. Increased NF-kB DNA-binding activity in nuclear extracts prepared from the ischemic hemisphere was further substantiated by electrophoretic mobility gel-shift analysis. Because the activation of NF-kB by many stimuli can be blocked by antioxidants in vitro, the effect of the antioxidant, LY341122, previously shown to be neuroprotective, on NF-kB activation in the MCAO model was evaluated. No significant activation of NF-kB was found by electrophoretic mobility gel-shift analysis in animals treated with LY341122. These results demonstrate that transient focal cerebral ischemia results in activation of NF-kB in neurons and supports previous observations that neuroprotective antioxidants may inhibit neuronal death by preventing the activation of NF-kB.
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Oliveira, Vasco A., Linda Mathews, Danielle Yarde, Xingyu Wang, David Boulware, Lori A. Hazlehurst, Dung-Tsa Chen, Amer Beg, and William S. Dalton. "NF-kB as a Regulator of FA/BRCA Gene Expression in Multiple Myeloma." Blood 110, no. 11 (November 16, 2007): 3508. http://dx.doi.org/10.1182/blood.v110.11.3508.3508.
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Abstract Results to date argue compellingly that disruption of FA/BRCA gene expression plays a pivotal role in human somatic carcinogenesis. Melphalan, a DNA cross-linker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). Although most patients respond to standard and high dose melphalan, eventually patients acquire resistance and develop progressive disease. In 1991, our laboratory reported that acquired resistance in a human myeloma cell line was associated with reduced DNA crosslinks, elevated glutathione levels, and increased radiation survival (Cancer Res. 5:993; 1991). Most recently, we reported that the melphalan-resistant myeloma cell lines, 8226/LR5 and U266/LR6, showed a significant increase in several FA/BRCA genes compared to drug-sensitive cells, and that enhanced interstrand crosslink (ICL) repair via this signaling pathway contributes to acquired drug resistance in melphalan resistant cell lines (Blood 10:698; 2005). Here, we report that IKKa is constitutively phosphorylated in unstimulated 8226/LR5 cells, but not in melphalan-sensitive control cells. The specific phosphorylation of IKKa leads to an increase in basal NF-kB DNA binding activity, and 8226/LR5 cells are found to be markedly sensitive to BMS-345541 (a highly selective inhibitor of IkB) relative to control cells. Importantly, a cytotoxic dose of BMS-345541 induces a dramatic decrease in FA/BRCA gene expression, and a concomitant inhibition of NF-kB DNA binding activity in both 8226/S and 8226/LR5 cells. Furthermore, we show that 8226/LR5 cells experience the highest degree of direct binding between FANCD2 promoter and NF-kB/Rel family members, which, in turn, leads to an increase in basal FANCD2-specific NF-kB activity. Small-interfering RNA (siRNA)-mediated depletion of RelB and p50, but not other NF-kB subunits, in 8226 cells results in impaired NF-kB binding activity, and visible decrease in FANCD2 protein expression. Studies designed to dissect the role of NF-kB in acquired melphalan resistance are in progress, and the results will be presented. Our findings suggest that NF-kB functions as a regulator of FA/BRCA expression, and that this pathway represents a new target for preventing acquired drug resistance in myeloma patients.
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Kurniawan, Shahdevi Nandar, Muthia Marwah, Rodhiyan R, and Nanik S. "THE EFFECT OF 500 NM DOSAGE OF NIMODIPINE ON NRF2 AND NF-KB EXPRESSION IN NEURON CELL LINE CULTURE SH-SY5Y EXPOSED WITH CHRONIC HYPERGLYCEMIA." International Journal of Research -GRANTHAALAYAH 8, no. 2 (May 30, 2020): 235–42. http://dx.doi.org/10.29121/granthaalayah.v8.i2.2020.214.
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Introduction: Metabolic stress and mitochondrial dysfunction in chronic hyperglycemia can cause an increase in Ca2+ cytosol ions that cause dysfunction of NRF2, that will increase the activity of NF-kB which causes an increase in cytokine production, where NF-kB can modulate NRF2 transcription and its activity, so that it has positive or negative effects in the target gene. Giving nimodipine in chronic hyperglycemia conditions can increase levels of NRF2 molecules and reduce levels of NF-kB. Aim: The purpose of this research is to know the effect of treating nimodipine dose 5 μM towards expression of the protein NRF2and NF-kB in SH-SY5Y cell culture induced by chronic hyperglycemia.
Method: This study is a true experimental study using randomized posttest only controlled group design in human SH-SY5Y neuron cell culture. Neuron cells were exposed with chronic glucose (25 mM / G25 normoglycemia, 50 mM / G50 hyperglycemia) for 6 days, followed by administration of nimodipine (without nimodipine and nimodipine 500 nM) for 30 minutes. Calculation of NRF2 and NF-kB expressions done by fluorescence method.
Results: There were significant differences in the NF-kB neuron cells in the treatment group with controls, both normoglycemia and hyperglycemia (p <0.05). Nimodipine was not able to increase the expression of NRF-2 in the treatmnrnt group, although there were significance differences between these two groups. The Spearman Rank correlation test showed a negative correlation between NRF2 and NF-kB expression in the SH-SY5Y human cell line neuron culture that exposed to chronic hyperglycemia with administration of 500 nM nimodipine.
Conclusion: There was a statistically significant decrease in NF-kB expression in SH-SY5Y neuron cells that exposed to chronic hyperglycemia given nimodipine compared to controls group. Nimodipine treatment to SH-SY5Y neuron cells exposed to hyperglycemia not yet increase expression of NRF2.
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Allen, Kristi L., Mukesh K. Jain, and Keith R. McCrae. "Decreased Expression of KLF2 and KLF4 Induced by Antiphospholipid Antibodies Promotes NF-Kb-Mediated Endothelial Cell Activation and Is Modulated by CBP/p300." Blood 116, no. 21 (November 19, 2010): 4315. http://dx.doi.org/10.1182/blood.v116.21.4315.4315.
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Abstract Abstract 4315 Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy loss in the presence of antiphospholipid antibodies (APLA). These antibodies are directed primarily against phospholipid-bound β2-glycoprotein I (β2GPI). Anti-β2GPI antibodies activate endothelial cells, enhancing the expression of adhesion molecules and tissue factor, and the secretion of proinflammatory cytokines. Krüppel-like factors (KLF) regulate endothelial cell inflammatory responses. KLF2 and KLF4 mediate anti-atherosclerotic and anti-inflammatory effects in endothelial cells, and we have hypothesized that alterations in the expression or activity of KLF2 or KLF4 may modulate the endothelial cell response to APLA. In preliminary studies, we have observed that endothelial cell activation induced by APLA/anti-β2GPI antibodies inhibits the expression of KLF2 and KLF4, and as demonstrated by our laboratory and others, is accompanied by activation of NF-kB. However, forced expression of KLF2 or KLF4 by plasmid-mediated transfection of endothelial cells inhibits neither the phosphorylation of ser536 of the p65 subunit of NF-kB, nor the nuclear translocation of p65 in response to APLA/anti-β2GPI antibodies. Despite the lack of effect on forced KLF2 or KLF4 expression in endothelial cells on p65 phosphorylation, expression of either of these factors inhibits NF-κB transcriptional activity with corresponding inhibition of cellular activation as measured by inhibition of cell-surface E-selectin expression as well as E-selectin promoter activity. Inhibition of NF-kB transcriptional activity by KLF2 and KLF4 appears to be due to recruitment of the CBP/p300 cofactor away from NF-kB by KLF2 or KLF4, since augmenting the cellular pool of CBP/p300 by transfection restores NF-κB activity and endothelial cell activation responses. Similarly, treatment of APLA-activated endothelial cells with CBP/p300 siRNA inhibits NF-kB transcriptional activity regardless of the levels of KLF2 or KLF4. These data suggest that APLA inhibit KLF expression and that these changes promote the acquisition of a prothrombotic endothelial cell phenotype. CBP/p300 may serve as a molecular switch that determines the relative antithrombotic activities of KLFs versus the prothrombotic, inflammatory responses induced by NF-kB in APLA/anti-β2GPI antibody activated endothelial cells. Disclosures: No relevant conflicts of interest to declare.
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Huang, Fei, Li-Yin Hung, and Reen Wu. "The differential roles of Act-1 and NF-κB in the regulation of various IL-17A-induced genes in A549 cells (134.95)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 134.95. http://dx.doi.org/10.4049/jimmunol.182.supp.134.95.
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Abstract It has been shown that IL-17A is a potent inducer of human beta-defensin (HBD)-2 in airway epithelia through the Act1-dependent NF-kB transcriptional regulation. In our recent findings, the induction of HBD-2 is very robust in human primary airway epithelia and human bronchial epithelial-1 (HBE-1). In contrast, A549, an alveolar epithelial cell line, has a very weak IL-17A-stimulated HBD-2 gene expression. We found A549 cell has an impaired NF-kB activation specific to IL-17A cytokine, since other pro-inflammatory cytokines could stimulate significant NF-kB and HBD-2 gene expression. In addition, over-expression of Act-1 protein in HBE-1 can significantly induce HBD-2 promoter activity in the absence of IL-17A, while in A549, no HBD-2 induction was observed regardless of the presence of IL-17A. Interestingly, the IL-17A-stimulated genes that have been discovered so far are mostly dependent on Act-1 and NF-kB activation. However, we found the extent of the regulation by Act-1 and NF-kB varies between different genes. By use of siRNA knockdown, we found some IL-17A-induced genes are strictly regulated by Act1 but only partially by NF-kB. Therefore, IL-17A can stimulate certain gene expressions even under a deficient NF-kB activation in A549 cells. These findings suggested there might be some molecules in addition to NF-kB regulated by Act1 to stimulate IL-17A gene expression.
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Morotti, Alessandro, Veronica Ullmannova, Daniela Cilloni, Francesca Messa, Manuela Messa, Ilaria Defilippi, Francesca Arruga, et al. "Over-Expression of the P65 Subunit of NF-kB and Increased DNA Binding Activity of NF-kB Is a Common Event Both in Philadelphia Positive and Negative Chronic Myeloproliferative Disorders." Blood 104, no. 11 (November 16, 2004): 4759. http://dx.doi.org/10.1182/blood.v104.11.4759.4759.
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Abstract The chronic myeloproliferative diseases (CMPD) are clonal disorders characterized by increased proliferation of cells from one or more myeloid lineages. The most common CMPD is Chronic Myeloid Leukemia which is characterized by the Philadelphia t(9;22) chromosomal translocation. The pathogenesis of Philadelphia negative CMPD is poorly understood, although the activation of tyrosine kinases appears to be an essential feature. For example, a constitutively activated PDGF receptor tyrosine kinase (FIP1L1-PDGFRA) is involved in some cases of the hypereosinophilic syndrome (HES), which is a form of CMPD characterized by increased proliferation of eosinophils. Different reports have demonstrated that the transcription factor NF-kB is essential for Bcr-Abl mediated transformation. NF-kB is a transcription factor which is composed of two subunits (generally p65 and p50). NF-kB dimers are retained into the cytoplasm by the inhibitory protein IkB. Different stimuli trigger the Serine phosphorylation of IkB and its proteolitc degradation. Free NF-kB translocates into the nucleus where it mediates the transcription of different genes involved in cellular proliferation, transformation and in apoptosis resistance. The aim of this work is to evaluate whether NF-kB is active both in Ph positive and in Ph negative CMPD. Bone marrow samples of 8 myeloproliferative disorders (3 Philadelphia positive CML, 3 Ph negative CML-like, 1 HES, 1 Idiopathic Myelofibrosis) have been collected at the diagnosis. The t(9;22) positive K562 cell line, derived form a CML blast crysis, has been used as a positive cellular control of the following experiments. Each samples have been lysed to obtain cytosolic and nuclear extracts. Western blot have been performed to evaluate the expression of the p65 subunit of NF-kB, the regulatory protein IkB and the antiapoptotic protein Bcl-2, whose expression may be regulated by NF-kB. Subsequently the DNA binding activity of NF-kB have been measured with an ELISA method. Our data shows that in all samples p65 is over-expressed both in the cytosol and in the nucleus respect to normal peripheral blood and normal bone marrow samples. The antiapoptotic Bcl-2 is also detectable by western blot in all pathological samples. In normal samples IkB is detected only in the cytosol and not in the nucleus while in CMPD samples it is expressed predominately in the nucleus. Basal DNA binding activity of NF-kB is increased in all the nuclear samples but not in normal samples. To assess whether NF-kB is directly involved in the control of cell growth and apoptosis, we have developed a stable K562 cell line expressing the super repressor IkB. The SR-IkB is a mutated for of IkB which can not be degraded causing an cytosolic sequestration of NF-kB. This stable cell line has a marked reduction of cell growth and is more sensible to the apoptotic stimuli. The data described above suggest that NF-kB activation may be a common mechanism of transformation in both Ph positive and negative CMPD and that its inhibition may be a powerful targeted molecular therapy.
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Gu, Lubing, Ningxi Zhu, Harry W. Findley, and Muxiang Zhou. "Loss of PTEN Expression Induces NF-kB Via PI3K/Akt Pathway Involving Resistance to Chemotherapy in Acute Lymphoblastic Leukemia Cell Lines." Blood 104, no. 11 (November 16, 2004): 4438. http://dx.doi.org/10.1182/blood.v104.11.4438.4438.
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Abstract PTEN is a tumor suppressor gene responsible for downregulating the phosphoinositide 3-kinase (PI3k)/Akt pathway. Loss of PTEN expression frequently occurs in human cancer leading to high Akt activation, which consequently confers neoplastic cell survival and resistance to chemotherapy-induced apoptosis. Here we report a mechanism by which loss of PTEN expression activates the transcription factor NF-kB through the PI3k/Akt pathway that induces activation of the IkBa kinase (IKK). Activation of NF-kB by loss of PTEN expression results in resistance to doxorubicin in acute lymphoblastic leukemia (ALL) cells. Initially, we examined 27 leukemia cell lines derived from children with ALL for the expression of PTEN and constitutive activation of NF-kB to evaluate whether there is a correlation between these two events. We found that 12 of the 27 lines lacked PTEN expression (PTEN-). Of 12 PTEN- ALL lines, 10 lines expressed constitutive NF-kB activation. In contrast, 11 of the 15 PTEN positive (PTEN+) lines were defect of NF-kB activation. Treatment of PTEN- line with PI3k kinase inhibitor Ly294002 caused downregulation of Akt activity accompanied by reduced activation of IKK and inhibition of constitutive NF-kB activation, resulting in increased sensitivity to doxorubicin-induced apoptosis. Similar to treatment with Ly294002, transfection of the PTEN expression plasmid into the PTEN- lines attenuated constitutive activation of both Akt and NF-kB, thereby sensitizing these cells to doxorubicin. These results suggest that both constitutive and inducible activation of NF-kB play an important role in chemotherapy resistance, and that loss of PTEN expression is at least one reason for the constitutive activation of NF-kB in ALL cells.
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Qu, Changju, Yadong Liu, Kranthi Kunkalla, Nitin K. Agarwal, and Francisco Vega. "Smoothened (SMO) Activates NF-Kb Pathway Through Activation of PKCβ/CARMA1 and TRAF6 Stabilization in Diffuse Large B-Cell Lymphoma." Blood 120, no. 21 (November 16, 2012): 1298. http://dx.doi.org/10.1182/blood.v120.21.1298.1298.
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Abstract Abstract 1298 Aberrant activation of hedgehog (Hh) and NF-kB pathways contribute to tumor cell growth, survival and chemotolerance in diffuse large B-cell lymphoma (DLBCL). Previously, we documented a functional crosstalk between hedgehog (Hh) and NF-kB pathways that contribute to tumor cell growth and survival in diffuse large B-cell lymphoma (DLBCL). However, the molecular mechanisms that link Hh with NF-kB pathway have not been defined. Based on that smoothened (SMO) has been associated with heterotrimeric G protein members of the Gα i family and established as a GPCRs-like protein, we hypothesize that GPCRs-related mechanisms such as the protein kinase C (PKC)-CARMA1-BCL10/MALT1/TRAF6 axis may contribute to SMO-dependent activation of NF-kB. First, we confirmed that SMO contributes to the activity of total PKC in 293T and DLBCL cells. Transiently or stably silencing of SMO resulted in decreased total PKC activity in comparison with the controls, while overexpression of SMO resulted in increased activity of PKC. Cyclopamine-KAAD or recombinant Shh N-terminal peptide resulted in decrease or increase of the total activity of PKC, respectively. As PKC isoforms β-1 and β-2 are the major isoforms expressed in B-lymphocytes that mediates NF-kB activation induced by activation of the B-cell receptor, we assessed the activation status of these two isoforms in response to changes of SMO activity. Inhibiting SMO with cyclopamine-KAAD or silencing SMO by siRNA decreased the phosphorylation status of PKCβ-1 and −2. In contrast, activating SMO with Shh N-terminal peptide increased the phosphorylation of PKCβ-1 and −2. Next, we assessed if SMO can modulated the activity of CARMA1. CARMA1 is a scaffold protein that serves to integrate the upstream signal of PKCs with downstream effectors in hematopoietic cells. We found that cyclopamine-KAAD or silencing SMO by siRNA decreased the phosphorylation status (inactivation) of CARMA1 and that activation of SMO with Shh N-terminal peptide increased the phosphorylation (activation) of CARMA 1. Because the polyubiquitination of TRAF6 and NEMO (IKKg) at lysine 63 (K63) are important events in propagating NF-kB signaling we examined the effect of overexpressing SMO on K63 polyubiquitination of TRAF6 and NEMO. Overexpression of SMO resulted in increased polyubiquitination of TRAF6 and NEMO at K63 supporting NF-kB pathway activation. TRAF6 is involved in activation of TAK1 and the IKK complex, resulting in translocation of NF-kB to the nucleus and activation of NF-kB. Silencing SMO by shRNA decreased total levels of TRAF6 that was associated with faster proteosomal degradation of TRAF6 in 293T cells. Inhibition of SMO with cyclopamine-KAAD also decreased the total levels of TRAF6 in DLBCL cells. In addition, overexpression of SMO (including a constitutively active mutated SMO) in 293T cells caused increase of TRAF6 expression by decreased polyubiquitination of TRAF6 at lysine 48 (K48) that targets TRAF6 for proteosomal degradation. In summary, altogether, these findings support that at least one mechanism by which SMO contributes to modulate activation of NF-kB is through the activation of the axis PKCβ/CARMA1/TRAF6/NEMO and stabilization and blocking degradation of TRAF6. Disclosures: No relevant conflicts of interest to declare.
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OJAYM, ALHUSSAIN A., Bing Yu, and Min-Ho Kim. "Fenton-catalytic nanocomposites for reprograming macrophages towards the improved anti-biofilm activity." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 86.9. http://dx.doi.org/10.4049/jimmunol.204.supp.86.9.
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Abstract Bacterial infections involving the formation of biofilms are associated with diminished bactericidal activity of macrophages, which has been shown to contribute to the persistence in chronic infection. We recently reported that S. aureus biofilm-induced impairment of bactericidal activities of macrophages are associated with attenuation of reactive oxygen species (ROS) generation and NF-κB activation. The objective of this study is to validate the use of biocompatible Fenton-catalytic nanocomposites composed of iron oxide nanoparticles (IONPs), a reducing agent (ascorbic acid), and hydrogen peroxide (H2O2) to take advantage of their ability to stimulate the generation of ROS and activation of NF-kB in macrophages by means of triggering a Fenton reaction. Using an in vitro culture model of NF-kB reporter-RAW 264.7 macrophages, we optimized the range of the concentrations of IONPs, ascorbic acid, and/or H2O2, along with their respective combinations, exhibiting an enhanced NF-kB activity and ROS generation. Based on the findings, we subsequently validated the effect of nanocomposite on the bactericidal activity of macrophages exposed to S. aureus biofilm. Our results revealed that RAW 264.7 cells treated with IONPs resulted in significant reduction in the number of surviving bacteria by ~50% and its efficacy further increased in combination with ascorbic acid and/or H2O2, which were in line with the extent of NF-kB activation and ROS generation. In summary, our study supports that harnessing the characteristic of Fenton-catalytic nanocomposite to tune macrophage polarization to exhibit a bactericidal activity may provide a new strategy for treating infectious diseases. Funding support: Supported by NIH R01 NR 015674
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Geng, Jian-Guo, Jian-Guo Wang, Nigel Mackman, Arne Slungaard, Yuqing Huo, and Nigel S. Key. "Regulation of Tissue Factor by NF-kB Transcription Factor p50 Is Essential for the Pathogeneses of Deep Vein Thrombosis and Arterial Restenosis." Blood 108, no. 11 (November 16, 2006): 1458. http://dx.doi.org/10.1182/blood.v108.11.1458.1458.
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Abstract NF-kB transcription factors regulate the expression of tissue factor (TF), a principal initiator for the coagulation cascade. Dominant among the five cellular members of NF-kB transcription factors is the p50/p65 heterodimer. Here we report that Andrographolide (Andro; a 350-dalton antagonist that targets reduced cysteine62 of p50 for inhibition of NF-kB activation) and genetic deletion of p50 potently attenuated TF activity in stimulated endothelial cells and monocytes/macrophages. The direct binding of p50/p65 heterodimer to the TF-kB site in human TF promoter was demonstrated by p50 and p65 antibody ‘supershift’ using electrophoretic mobility shift assay and immunoprecipitation of the promoter of the human TF gene from chromatins of TNF-a-stimulated human umbilical vein endothelial cells. Andro-treated and p50 null mice both exhibited suppressed TF expression, blunted fibrin deposition, reduced venous thrombosis, and decreased neointimal hyperplasia. Blockade of TF activity by an anti-murine TF antibody also attenuated venous thrombosis and neointimal proliferation in vivo. Our findings thus indicate that NF-kB transcription factor p50 critically regulates TF activity in the pathogeneses of deep vein thrombosis and arterial restenosis, and suggest that specific inhibitors of p50, such as Andro, have the potential to be therapeutically valuable for preventing and perhaps treating arterial and venous thrombosis.
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Hideshima, Teru, Noopur Raje, Ruben Carrasco, Hiroshi Ikeda, Yutaka Okawa, Tanyel Kiziltepe, Sonia Valet, et al. "Delineation of Canonical and Non-Canonical NF-κB Pathways in Multiple Myeloma: Therapeutic Implications." Blood 110, no. 11 (November 16, 2007): 670. http://dx.doi.org/10.1182/blood.v110.11.670.670.
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Abstract NF-κB pathway plays a crucial role in the pathogenesis in cancer cells including multiple myeloma (MM). The NF-κB complex is dimer in different combinations of Rel family proteins, including p65 (RelA), RelB, c-Rel, p50 (NF-κB1), and p52 (NF-κB2). Recent studies have revealed that NF-κB activity is mediated via two distinct pathways. In the canonical pathway, NF-kB is typically a heterodimer composed of p50 and p65 subunits. In the non-canonical pathway, NF-kB is typically a heterodimer composed of RelB and p100 subunits. We have shown anti-MM activities of IKKβ inhibitors (PS-1145, MLN120B); however, effects of these agents were modest. Our studies therefore suggest that baseline NF-kB activity in MM cells is not totally dependent on the canonical pathway, and that inhibition of only canonical NF-κB pathway may not be sufficient to block total NF-kB activity. In this study, we therefore hypothesized whether non-canonical inhibitors significantly enhanced NF-κB inhibition induced by canonical inhibitors in MM cells. We first examined baseline NF-κB activity using electrophoretic mobility shift assay (EMSA). NF-κB activity varied between cell lines; for example MM.1S, MM.1R and H929 cells have higher level of NF-κB activity than in RPMI8226, INA6 and OPM2 cells. To define the role of canonical and non-canonical pathway, we next examined protein expression of p50, p65 and p52 NF-κB in these cell lines: p65 was highly expressed in all MM cell lines; however, expression of p50 and 52 is variable. Surprisingly, no detectable or weak expression of p50 was observed in U266, RPMI8226, LR5, H929 and OPM2 cell lines, suggesting that baseline NF-kB activity in these cell lines is not maintained only by the canonical pathway. We then attempted to block non-canonical NF-κB pathway in MM cell lines. Specifically since IKKα and IKKβ are client proteins of heat shock protein (Hsp) 90, we examined whether 17AAG could inhibit expression and/or function of IKKα and IKKβ in MM cells. Importantly, both IKKα and IKKβ were significantly downregulated by 17AAG in MM cell lines. To determine whether downregulation of these IKK proteins by 17AAG was due to inhibition of transcription, we next performed real-time quantitative PCR and no significant inhibition of relative expression of IKKβ was observed by 17AAG treatment, suggesting that downregulation of these proteins was a post transcription event. We further examined whether 17AAG enhanced the effect of IKKβ inhibitor MLN120B on NF-κB activity. Although the inhibitory effect by either MLN120B or 17AAG alone on phosphorylation (p) of IκBα triggered by TNFα was marginal, combination treatment of MLN120B with 17AAG almost completely blocked IκBα, suggesting that this combination synergistically inhibit canonical NF-κB activity in MM cells. Importantly, the combination of MLN120B with 17AAG also significantly blocked baseline and TNFα-triggered NF-κB activity, assessed by EMSA, in MM cells. Finally, 17AAG augmented the growth inhibitory effect of MLN120B in the context of bone marrow stromal cells. Taken together, these results showed that baseline and TNFα-triggered NF-κB activities were completely blocked by this combination treatment, and provide the rationale for its clinical evaluation to induce maximum inhibition of NF-κB activity and improve patient outcome in MM.
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Lenz, Georg, R. Eric Davis, Vu N. Ngo, Lloyd T. Lam, Thaddeus George, George W. Wright, Sandeep S. Dave, et al. "CARD11 as an Oncogene in Diffuse Large B Cell Lymphoma." Blood 110, no. 11 (November 16, 2007): 692. http://dx.doi.org/10.1182/blood.v110.11.692.692.
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Abstract The activated B-cell-like (ABC) molecular subtype of diffuse large B cell lymphoma (DLBCL) is characterized by constitutive activation of the nuclear factor-kB (NF-kB) pathway, which it requires for survival. By contrast, the NF-kB pathway is infrequently activated in the germinal center B cell-like (GCB) subtype of DLBCL. A crucial protein involved in activation of the NF-kB pathway by antigen receptor stimulation in normal B and T lymphocytes is CARD11. A previous loss-of-function genetic screen using RNA interference identified the signaling pathway from CARD11 to IkB kinase b as essential for the constitutive NF-kB activity in ABC DLBCL. However, the oncogenic mechanisms underlying the activity of CARD11 in this lymphoma subtype have yet to be elucidated. To this end, we resequenced some or all of the CARD11 exons in 157 primary DLBCL patient samples and cell lines and, as a control, in 19 MALT lymphoma patient samples. Various missense mutations were discovered in 10.4% of ABC DLBCL samples (8/77), and all mutations were located in exons encoding the coiled-coil domain of CARD11. In contrast, CARD11 coiled-coil domain mutations were detected in only 3.8% of the GCB DLBCL patient samples (3/80) and in no MALT lymphoma samples. ABC and GCB DLBCLs with CARD11 mutations were characterized by high expression of an NF-kB gene expression signature. Experimental introduction of each CARD11 mutant form in lymphoma cell lines altered their NF-kB signaling properties. Six of the CARD11 mutants caused strong constitutive NF-kB activation in the absence of any exogenous stimulus, a phenotype that was not elicited by wild type CARD11. For other CARD11 mutants, the enhancement of NF-kB signaling was most apparent upon antigen receptor stimulation. Fluorescence microscopy of cells bearing GFP-tagged CARD11 mutants revealed one or more prominent cytosolic aggregates in most cells whereas GFP-tagged wild type CARD11 was distributed diffusely in the cytoplasm. Multispectral imaging flow cytometry (Imagestream, Amnis) was used to quantitate the degree of aggregate formation by the CARD11 mutants. This analysis revealed a positive correlation between aggregate formation and NF-kB pathway activation. Immunofluorescent staining revealed that endogenous IkB kinase proteins were co-localized with the CARD11 mutants in the cytosolic aggregates, supporting the hypothesis that aggregate formation plays a central role in the activation of NF-kB by the CARD11 mutants. These results demonstrate that CARD11 is a bone fide oncogene in DLBCL. Mutations in the CARD11 coiled-coil domain caused spontaneous multimerization of the protein leading to IkB kinase recruitment and NF-kB signaling, thereby preventing cell death. These findings provide a strong genetic rationale for the development of CARD11 pathway inhibitors for the therapy of DLBCL.
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Péant, B., J. Diallo, L. Lessard, A. Mes-Masson, and F. Saad. "Regulation of IKKe expression by androgen receptor and NF-kB transcriptional factor in prostate cancer." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 10106. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.10106.
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10106 Background: In unstimulated cells, NF-kB transcription factor is sequestered in the cytoplasm as an inactive p65/p50 dimer through interaction with a member of the inhibitor of kB protein family (IkBa). Prominent constitutive activation of NF-kB was observed in prostate cancer (PCa) cell lines lacking androgen receptor (AR) expression (PC3 and DU145) whereas only very low levels of NF-kB activity were seen in androgen-dependent cell lines (LNCaP and CWR22Rv1). As IkB kinase-e (IKKe) has recently been shown to be controlled by NF-kB, we hypothesize that IKKe may be involved in PCa progression based on its interaction with the NF-kB protein, and that these interactions are influenced by AR signaling. Methods: LNCaP cells were used to study IKKe expression with or without stimulation by the analog of androgen R1881 and by the tumor necrosis factor (TNF)-a. IKKe protein and RNA expression were characterized by immunoblot assay and quantitative PCR, respectively. IKKe expression was then correlated with p65 nuclear localisation. NF-kB activity was inhibited using an IkBa dominant negative construction. Inhibition of AR synthesis was performed using a siRNA against AR. Results: IKKe gene expression was stimulated by TNF-a treatment in LNCaP cells and inhibited by transfection of a dominant negative form of IkBa which prevented the nuclear translocation of p65. We also observed constitutive IKKe expression in hormone-refractory cells. Furthermore, we showed that TNF-a-induced IKKe expression is inhibited by R1881 in hormone-responsive PCa cells and this inhibition was correlated with the modulation of IkBa expression by R1881. Finally, we observed that the expression of IKKe is constitutively induced after blocking AR expression in LNCaP cells. Conclusions:. Our results show that IKKe expression is regulated by NF-kB in PCa cell lines. Moreover, IKKe appears to be down-regulated by ligand-dependent AR signaling through the control of IkBa expression. Further studies will be needed in order to determine the implications of this phenomenon with regard to NF-kB regulation, androgen resistance and effect on PCa progression. No significant financial relationships to disclose.
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Roh, Eunmiri, Heun-Sik Lee, Jeong-Ah Kwak, Sang Hun Jung, Sang-Bae Han, and Youngsoo Kim. "Novel chalcone JSH 4-4 inhibit NF-kB-regulated inflammatory gene expression via targeting LPS-binding site of MD-2 (98.4)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 98.4. http://dx.doi.org/10.4049/jimmunol.184.supp.98.4.
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Abstract Previous studies have shown that chalcone has beneficial effects with antioxidant and anti-inflammatory activities. However, molecular basis for these effects not yet fully understood. In this study, a chalcone derivative, 2’, 4’-dihydroxy-6’-isopentyloxy chalcone (JSH 4-4) was discovered as an inhibitor of NF-kB activation. To demonstrate molecular target affected by JSH 4-4, we examined a homotypic interaction of TLR4, using Ba/F3 cells stably expressing TLR4-Flag, TLR4-GFP CD14 and MD-2, seperately. As a result, JSH 4-4 inhibited the LPS-induced TLR4 oligomerization. Moreover, we demonstrated that JSH 4-4 inhibit LPS-induced NF-kB activation via targeting LPS-binding site of MD-2. This mechanism of action could contribute to suppressive effect of JSH 4-4 on NF-kB-regulated production of inflammatory mediators such as nitric oxide, COX-2 and cytokines. TLR4-mediated NF-kB signalling occurs following sequential activation of IRAK family and TAK1. JSH 4-4 inhibited LPS-induced phosphorylation of IRAK-1 and TAK1. In addition, JSH 4-4 significantly inhibited IKK activity and phosphorylation of IkBa, resulting in sequential prevention of downstream events, including degradation of IkBs, nuclear translocation of NF-kB p65. In conclusion, JSH 4-4 inhibit NF-kB-regulated inflammatory mediators via targeting the receptor complex MD2-TLR4 in macrophages, thus JSH 4-4 might be considered as a potential agent for the treatment of NF-kB-associated inflammatory diseases.
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Arai, Ayako, Mayumi Takahashi, Ken-Ichi Imadome, Takatoshi Koyama, Yasunori Saitoh, Shoji Yamaoka, Shigeyoshi Fujiwara, and Osamu Miura. "NF-κB Is Constitutively Activated in EBV-Infected T or NK Cells and Can Be a Molecular Target for EBV-Positive T/NK-Cell Lymphoproliferative Disease Treatment." Blood 116, no. 21 (November 19, 2010): 1998. http://dx.doi.org/10.1182/blood.v116.21.1998.1998.
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Abstract Abstract 1998 Epstein-Barr virus (EBV) can infect not only B cells but also rarely T or NK cells and causes EBV-positive T/NK-cell lymphoproliferative disease (EBV-T/NK-LPD), such as extranodal NK/T-cell lymphoma (ENKL), aggressive NK-cell leukemia, and chronic active EBV infection (CAEBV). However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of these EBV-induced malignancies have not been elucidated to date. Furthermore, optimal chemotherapy for these, especially for CAEBV, has not been established and the prognosis of EBV-T/NK-LPD remains very poor. NF-kB is a transcription factor that mediates anti-apoptotic molecular signaling and promotes proliferation of cancer cells. NF-kB is known to be constitutively activated in some hematological malignancies; the proteasome inhibitor bortezomib suppresses its activity and is used clinically as an anticancer reagent. To clarify the molecular mechanism underlying the development of EBV-T/NK-LPD, we focused on NF-kB in four EBV-positive T- and NK-cell lines, SNT8, SNT15, SNT16, and SNK6, which had been established from primary lesions of ENKL patients (SNT8 and SNK6) and the peripheral blood (PB) of a CAEBV patient (SNT15, 16), respectively. In these cells, it was demonstrated that p50, p52, RelA, and RelB—the components of NF-kB—existed constitutively in the nucleus. On the other hand, the EBV-negative T-cell lines, Jurkat and Molt4, and an NK-cell line, KHYG1, were negative for nuclear localization of these molecules. Elevated nuclear NF- kB–DNA binding activity was demonstrated by electrophoretic mobility shift assay (EMSA), using oligonucleotides encoding an NF-kB-binding sequence as a probe. Supershift EMSA revealed that NF- kB–DNA binding complexes in these cells involved p50, p52, and RelA. In addition, reporter assay using NF-kB-dependent luciferase reporter gene consisting of an NF-kB-binding site as a promoter demonstrated constitutive activation of NF-kB in SNT8 cells, whereas not in Jurkat cells. From these results, we concluded that NF-kB was constitutively activated through the canonical and noncanonical pathways in these EBV-infected T- or NK-cell (EBV-T/NK cell) lines. Next, we investigated NF-kB activation in EBV-T/NK cells derived from CAEBV patients. CAEBV was diagnosed according to the following criteria: presence of persistent infectious mononucleosis-like symptoms, elevation of EBV-DNA titer in PB, and detection of EBV-infected T or NK cells with clonal proliferation. To detect infected cells, we isolated peripheral mononuclear cells and divided them into CD19-, CD4-, CD8-, or CD56-positive fractions using antibody-conjugated magnetic beads. The EBV DNA of each fraction was quantified using a real-time quantitative polymerase chain reaction assay. Ten patients (aged 8–72 years; 4 male, 6 female; infected cell types CD4:3, CD8:3, γδ:1, and CD56:3) were diagnosed as CAEBV. Then, we examined NF-kB activation in these cells. Similar to the cell lines, immunoblotting and EMSA demonstrated evidence of constitutive activation of NF-kB such as nuclear localization and DNA binding of p50, p52, and RelA in EBV-T/NK cells from CAEBV patients. To investigate the molecular mechanism of NF-kB activation in EBV-T/NK cells, we examined the influence of viral proteins expressed in EBV-T/NK cells, including LMP1, LMP2A, LMP2B, and EBNA1 (latency type 2). We performed luciferase reporter assay using expression vectors for these proteins in Jurkat cells. LMP1, most significantly, and LMP2A, to a lesser extent, upregulated NF-kB-dependent reporter gene expression in Jurkat cells, whereas the other viral proteins did not, suggesting that LMP1 and LMP2A mediated NF-kB activation in T/NK cells. Finally we investigated the contribution of NF-kB to the development of EBV-T/NK-LPD. Bortezomib, in a dose-dependent manner, inhibited translocation of p50, p52, and RelA to the nucleus and reduced the expression of Bcl-XL, which was a transcriptional target of NF-kB in SNT8, SNK6, and CAEBV patients’ cells. In addition, it was also confirmed that bortezomib (0.5nM -5nM) suppressed the survival of and induced apoptosis of EBV-T/NK cells derived from 5 CAEBV patients. These results suggest that constitutive activation of NF-kB contributes to the development of EBV-T/NK-LPD and that the NF-kB signaling pathway can be an attractive molecular target for treatment. Disclosures: No relevant conflicts of interest to declare.
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Yu, Hong-Nu, Young-Rae Lee, Hyun-Jaung Shim, Myung-Kwan Han, Eun-Kyung Song, Jinny Park, and Jong-Suk Kim. "Tumor Necrosis Factor-a Enhances Human Leukemia Cell Differentiation Induced by DMSO, but Not Retinoic Acid." Blood 106, no. 11 (November 16, 2005): 3893. http://dx.doi.org/10.1182/blood.v106.11.3893.3893.
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Abstract One of the human leukemia treatment methods is to differentiate leukemia cells into mature cells. Because differentiated cells lose their proliferative and tumor-forming abilities, differentiation inducers may be useful for the treatment of leukemia. Differentiation of leukemia cells has been studied using HL60 cells, a human promyelocytic leukemia cell line, which can be differentiated into granulocyte-like or monocyte/macrophage-like cells by various pharmacological agents such as dimethyl sulfoxide (DMSO), retinoic acid and phorbol myristic acetate (PMA). We previously reported that nuclear factor - kB (NF-kB) activation plays the important role in DMSO-induced differentiation of HL60 cells. Thus, we hypothesized that NF-kB activators could enhance DMSO-induced differentiation of HL60 cells. Here we examine whether tumor necrosis factor-a (TNF-a), a potent NF-kB inducer, enhance DMSO-induced differentiation of HL60 cells. TNF-a was found to enhance HL60 cell differentiation induced by DMSO. CD11b, a differentiation marker, was increased in 0.5 % DMSO-treated cells compared to control cells. When TNF-a was added to the same condition, CD11b expression was further enhanced in a dose and a time dependent manners. We also found that nitro blue tetrazolium (NBT) reducing activity, a marker for granulocytic differentiation, was further increased in DMSO plus TNF-a treated cells compared to only DMSO- treated cells. However, TNF-a alone had no effect on CD11b expression and NBT reducing activity. The enhancement of DMSO-induced HL60 differentiation by TNF-a was offset by NF-kB inhibition. Interestingly, retinoic acid- induced differentiation of HL60 cells showed no enhancing effect of TNF--a on the differentiation. These findings indicate that TNF--a might affect only NF-kB dependent differentation of HL60 cells. Taken together, we demonstrated that TNF-a enhances DMSO-induced differentiation of HL60 cells by stimulating NF-kB activation. Our results suggest that NF-kB inducers such as TNF-a are useful for the treatment of leukemia in combination with DMSO.
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Radigina, T., A. Illarionov, D. Kuptsova, A. Potapov, S. Petrichuk, and A. P. Fisenko. "P423 NF-kB as a prognostic marker of response to biologic therapy in children with IBD." Journal of Crohn's and Colitis 14, Supplement_1 (January 2020): S386—S387. http://dx.doi.org/10.1093/ecco-jcc/jjz203.552.
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Abstract Background The transcription factor NF-kB is a regulator of innate and adaptive immunity through the activation of various pro and anti-inflammatory mediators. The aim of this study was to assess the prognostic value of changes in the level of NF-kB translocation in lymphocyte populations in response to infusion of TNF blockers in children with IBD. Methods There were examined 44 children with IBD (27-CD, 17-UC) and 20 conditionally healthy children aged 3–18 years. Patient status (exacerbation, remission) was assessed by the PUCAI (UC) and PCDIA (CD) indices. There were determined the percentage of cells with NF-kB translocation in populations CD3+CD4+ (Th), CD3+CD8+(Tc), CD3-CD19+ (B cells), CD3-CD16 / 56+(NK cells), CD3+CD4+CD161+ (Th17), CD3+CD4+CD25highCD127low (Tregs) with NF-kB translocation kit using flow cytometry ImageStreamX MKII (Amnis) before infusion of TNF blockers (infliximab, adalimumab), one day after infusion and after 6 months therapy. Statistical evaluation was performed using a nonparametric Mann–Whitney test. The results are presented as the median per cent of cells with NF-kB translocation (Me [Q 0.25-Q 0.75]). Results It was observed an increased level of NF-kB translocation in B-lymphocytes (Me 64 [53–81] – Me 38 [33–45]; p = 6 × 10−5), NK cells (Me 40 [33 -55] – Me 21 [18–26]; p = 4x10-4), Tc (Me 20 [15–24] – Me 16 [13–17]; p = 0.03), Th17 (Me 23 [21–28] – Me 18 [16–19]; p = 0.005) in children with exacerbation compared with remission. The level of NF-kB translocation in populations of lymphocytes during the period of clinical endoscopic remission did not differ from conditionally healthy children. The correlation between the level of NF-kB translocation and the number of cells in the studied lymphocyte populations was not found. In children with IBD after infusion of TNF blockers, a decrease the level of NF-kB translocation in NK cells was observed (Me 27.3 [23.5–39.1] – Me 17.7 [16.5–26.5]; p = 0,03) and an increase in Tregs (Me 18.9 [16.91–20.8] – Me 26.9 [19.4–31.9]; p = 0.0015). Conclusion The level of NF-kB translocation reflects the functional activity of major and small populations of lymphocytes. An increase in activity of Tregs in response to the administration of TNF blockers allows predicting a positive outcome from therapy over the next 6 months. If there is no Tregs reaction in response to the administration of TNF blockers, treatment tactics should be reassessed.
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Baek, Suk-Hwan, Bin Huang, and Hyeun Wook Chang. "RNF144b is a negative regulator in TLR2-mediated NF-kB activation." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 132.4. http://dx.doi.org/10.4049/jimmunol.196.supp.132.4.
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Abstract NF-κB regulates the expression of a various genes involved in diverse cellular processes including inflammation and immunity. Activation of NF-κB requires ubiquitination, a highly conserved and versatile modification that can regulate cell signaling through both proteasome dependent and independent mechanisms. Ubiquitination process is a representative of post-translational modification involved in NF-κB activation of TNFR and TLR signaling. We demonstrated that RNF144b, an E3 ubiquitin ligase, is important factor for the NF-κB regulation in macrophages upon stimulation with TNFa or TLR2 ligand. Treatment of TNFa or TLR2 ligand (Pam3CSK4) decreased the expression of RNF144b mRNA on macrophages. TLR2 ligand has a stronger effect than TNFa in the expression of RNF144b mRNA. Knock-down of RNF144b potentiated activation of NF-kB. And according to the increase of NF-κB activity, the formation of foam cells and the production of cytokines (IL-1β, IL-6, CCL2 and CCL3) following TLR2 stimulation increased. On the contrary, overexpression of RNF144b decreased NF-κB activity and cytokines production in the TLR2-stimuated macrophages. RNF144b binds and ubiquitinates TRAF6 and these effects are dependent of its E3 ligase activity. Mutation of Cys201 to Ala abolishes activity of RNF144b to activate TRAF6 ubiquitination and also partially attenuates NF-κB activity. Taken together, these results suggest that RNF144b stimulates NF-κB activity via TRAF6 ubiquitination and then produces inflammatory cytokines and chemokines, and eventually controls foam cell formation in the TLR2-mediated macrophages.
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Camara-Clayette, Valerie, Yves Lepelletier, Ivan Moura, William Vainchenker, Olivier Hermine, and Vincent Ribrag. "Canonical NF-kB Pathway Is Not Constitutively Activated and May Not Be the Major Bortezomib Target in Mantle Cell Lymphoma (MCL)." Blood 108, no. 11 (November 16, 2006): 2591. http://dx.doi.org/10.1182/blood.v108.11.2591.2591.
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Abstract Bortezomib, a proteasome inhibitor, has promising activity in MCL. Bortezomib mechanism of action is complex since multiple proteins and cellular pathways are regulated by proteasome degradation. By blocking the degradation of IKBa, the canonical NF-kB pathway is one of the major Bortezomib target in lymphoid malignancies such as multiple myeloma. NF-kB activation has not been extensively assessed in other lymphoid malignancies such as MCL. NF-kB complexes (heterodimers P50/P65) were assessed by EMSA assays in REC GRANTA 519, NCEB, JEKO, JUN and UPN1 MCL cell-lines. EBV was present in 3/6 cell lines. P50/P65 complexes were detected in all EBV positive cell lines and only in 1/3 EBV negative cell-lines (JEKO). No nuclear P65 protein was detected by immunofluorescence or Western-blot analysis in the 2 other negative EBV cell lines (REC and UPN1). NF-kB transcriptional activity was measured by a luciferase-based reporter gene assay. Spontaneous NF-kB activity was low in UPN1, REC and JEKO compared to GRANTA 519 EBV positive cell-line (3 to 4 time lower) but can be strongly activated (up to 10 fold) using MEKK cotransfection assay. This suggest that NF-kB is functional, but not constitutively activated. Stable infections with Migr1-IRES-GFP IkBM (dominant negative IKB) or empty vector, were performed in GRANTA 519 EBV positive as well as in UPN1 cell line. No differences in proliferation or apoptosis were observed in UPN1 stably infected with IkBM or with the empty vector but GRANTA 519 EBV positive showed increased apoptosis and proliferation inhibition when infected with IkBM. Likewise, no P50/P65 heterodimers complexes were detected by EMSA in 4 patients with MCL. In vitro assays showed that UPN1 and JEKO cell lines had comparable sensitivities to Bortezomib than Multiple myeloma cell lines reported in the literature (IC 50: 6 nM and 12 nM respectively). This was also true for the 3 patients lymphoma cells assessed in vitro (12 nM). Taken together these results suggest that canonical NF-kB activation pathway is not constitutively active in EBV negative MCL cell lines and patients samples and could be associated with EBV infection in some MCL cell-lines. Therefore, this strongly suggests that Bortezomib target other molecules in MCL.
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Schrader, Carsten, Wolfram Klapper, Dirk Janssen, Paul Riis, Peter Meusers, Guenter Brittinger, Jens U. Siebmann, Michael Kneba, and Reza Parwaresch. "Analysis of Nuclear Factor Kappa B in Mantle Cell Lymphoma." Blood 106, no. 11 (November 16, 2005): 4712. http://dx.doi.org/10.1182/blood.v106.11.4712.4712.
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Abstract Gene expression profiles revealed that proliferation associated genes are important prognostic factors in the clinical outcome in mantle cell lymphoma (MCL). Beside this well accepted markers also analysis of apoptotic proteins are now under investigation. We investigated immunohistochemically the expression of the apoptotic marker NF-kB in relation to the clinical course in 89 patients enrolled in two multicenter prospective trials. Biopsies were recut and stained with mandatory antibodies (CD20, CD5, CD3, CD23, cyclin D1) and NF-kB. The NF-kB expression was analyzed in three groups: negative, cytoplasmatic positive and nuclear positive (more than 1/HPF). The expression was compared with the overall survival data analyzed according to Kaplan and Meier. In 13 cases a negative NF-kB staining was detected. Fifty-seven cases were positive only in the cytoplasma and in 17 cases more than 1 cell per high power field showed nuclear activity. Patients with mantle cell lymphoma that had negative and cytoplasmatic positive expression had a median overall survival time of 35.7 months compared to 22.4 months for patients with a nuclear NF-kB expression. The Kaplan-Meier analysis showed a significant difference in the overall survival time (p=0.0121). The immunohistochemical detection of NF-kB in mantle cell lymphoma is possible and a tool to identify patients with a poor prognosis.
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Jain, H., N. Dhingra, T. Narsinghani, and R. Sharma. "Insights into the mechanism of natural terpenoids as NF-kB inhibitors: an overview on their anticancer potential." Experimental Oncology 38, no. 3 (September 22, 2016): 158–68. http://dx.doi.org/10.31768/2312-8852.2016.38(3):158-168.
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The transcription factor, nuclear factor kappa B (NF-kB) is one of the principal inducible protein in mammals known to control the gene expression in many critical physiological responses such as oxidative stress, inflammation etc. and has been shown to play an important role in the pathogenesis of cancer. Terpenoids are major constituents present in nutritionally used fruits, vegetables and different spices which possess various pharmacological action including anticancer activity. Various terpenoids, viz. monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids and polyterpenoids inhibit NF-kB signaling pathway through IkB phosphorylation, DNA binding, p65 translocation etc. Keeping in mind these facts, the present review revealed the anti-cancer potential of naturally occurring terpenoids highlighting their mechanism of NF-kB inhibition. This review also focuses on some of the naturally occurring terpenoids belonging to various chemical categories with potential inhibitory effects on NF-kB and their role in the treatment of cancer.