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Interleukin-1β (IL-1) and tumor necrosis factor-α (TNF) are important pro-inflammatory cytokines involved in the mediation of the immune response, inflammation, tissue repair, and tumor progression. Regulation of IL-1 and TNF expression is mediated at the level of transcription by the transcription factor NFκB. Inhibition of NFκB activity by the proteasome inhibitor bortezomib (BZ) has been used as a frontline therapy in multiple myeloma and other hematological malignancies. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the NFκB recruitment to endogenous IL-1 and TNF promoters in BZ-treated human macrophages. Corresponding to the BZ-suppressed mRNA levels of IL-1 and TNF, we show that BZ inhibits p65 NFκB recruitment to IL-1 and TNF promoters. This study specifically uses U937 macrophages, but the protocol could be easily modified to analyze the regulation of NFκB recruitment in other cell types.

Transcription factor NFκB is a key regulator of genes involved in immune and inflammatory responses, as well as genes regulating cell proliferation and survival. In addition to many inflammatory disorders, NFκB is constitutively activated in a variety of human cancers and leukemia. Thus, inhibition of NFκB DNA binding activity represents an important therapeutic approach for disorders characterized by high levels of constitutive NFκB activity. We have previously shown that NFκB DNA binding activity is suppressed by the nuclear translocation and accumulation of IκBα, which is induced by inhibition of the 26S proteasome. In this chapter, we describe a protocol that uses small inhibitory RNA (si RNA) interference followed by electrophoretic mobility shift assay (EMSA) to analyze the regulation of NFκB DNA binding by nuclear IκBα induced by the proteasome inhibitor MG132. Using this protocol, we show that in human leukemia Hut-78 cells that exhibit high levels of NFκB DNA binding activity, MG132 induces nuclear translocation and accumulation of IκBα, which then specifically inhibits NFκB DNA binding. This protocol uses human leukemia Hut-78 cells; however, it can be easily adapted for other cells exhibiting high levels of constitutive NFκB DNA binding.

Transcription factor NFκB comprises a family of proteins that serve as crucial regulators of genes involved in host immune and inflammatory responses, cell survival, proliferation, and differentiation. Since transcription of NFκB-dependent genes is increased in numerous inflammatory disorders as well as in many types of cancer and leukemia, inhibition of NFκB-dependent transcription thus represents an important therapeutic target. We have previously shown that in human leukocytes, transcription of NFκB-dependent genes is inhibited by the nuclear translocation and accumulation of IκBα, which can be induced by an inhibitor of CRM1-dependent nuclear export, leptomycin B (LMB). In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the regulation of NFκB recruitment to NFκB-dependent promoters by nuclear IκBα induced by LMB. We show that in lipopolysaccharide (LPS)-stimulated human U-937 macrophages, recruitment of NFκB p65 and p50 proteins to NFκB-dependent promoters of IκBα and cIAP2 genes is suppressed by the LMB-induced nuclear IκBα. Even though in this study we use U-937 macrophages, this protocol should be readily adaptable to analyze the regulation of NFκB recruitment by nuclear IκBα also in other cell types.

Cutaneous T-cell lymphoma (CTCL) is characterized by constitutive activation of nuclear factor κB (NF-κB), which plays a crucial role in the survival of CTCL cells and their resistance to apoptosis. NF-κB activity in CTCL is inhibited by the proteasome inhibitor bortezomib; however, the mechanisms remained unknown. In this study, we investigated mechanisms by which bortezomib suppresses NF-κB activity in CTCL Hut-78 cells. We demonstrate that bortezomib and MG132 suppress NF-κB activity in Hut-78 cells by a novel mechanism that consists of inducing nuclear translocation and accumulation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), which then associates with NF-κB p65 and p50 in the nucleus and inhibits NF-κB DNA binding activity. Surprisingly, however, while expression of NF-κB-dependent antiapoptotic genes cIAP1 and cIAP2 is inhibited by bortezomib, expression of Bcl-2 is not suppressed. Chromatin immunoprecipitation indicated that cIAP1 and cIAP2 promoters are occupied by NF-κB p65/50 heterodimers, whereas Bcl-2 promoter is occupied predominantly by p50/50 homodimers. Collectively, our data reveal a novel mechanism of bortezomib function in CTCL and suggest that the inhibition of NF-κB-dependent gene expression by bortezomib is gene specific and depends on the subunit composition of NF-κB dimers recruited to NF-κB-responsive promoters.

Controlled nucleocytoplasmic localization regulates activity of NF kappa B as well as other transcription factors. Analysis of the nucleocytoplasmic protein shuttling has been greatly facilitated by the use of leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export. The authors have previously shown that LMB inhibits NF kappa B activity in human neutrophils by increasing the nuclear accumulation of NF kappa B inhibitor, I kappa B alpha. In this chapter, the authors describe a protocol that uses LMB to study the nucleocytoplasmic shuttling of I kappa B alpha in human macrophage-like U937 cells, thus inhibiting NF kappa B activity. This protocol should be readily adaptable to analyze the nucleocytoplasmic shuttling of other proteins in human leukocytes.

The objective of this study was to test the hypothesis that in an animal model of cathartic-induce intestinal dysfunction the proabsorptive effects of gum arabic (GA) could be associated with modulation of nuclear factor-kappaB (NF-kappaB) and with reduction of the inflammatory response caused by cathartics, as evidenced by intestinal mucosa cytokine production and gene expression. Juvenile male rats were given a phenolphthalein-magnesium citrate solution for 6 days, by itself or supplemented with either 10 or 20 g L(-1) GA, as a sole source of fluid. The controls given were tap water alone or with added 20 g L(-1) GA. The animals were euthanized and small-intestinal mucosa nuclear fractions and RNA were isolated. NF-kappaB p65 activity was highest after administration of cathartics, lowest in controls, and intermediate in GA-treated rats. Mucosal IL-1beta was overexpressed in tissues from cathartic-treated rats and from rats given high-GA solutions. Gene-array analysis revealed a complex pattern of gene regulation by cathartics which selectively upregulated several subfamilies of cytochrome P-450 family 2 genes. Co-administration of GA did not block this effect. These findings suggest that local anti-inflammatory effects on the small intestine could be obtained by administration of a nonabsorbable proteoglycan such as GA.

Increased activation of the transcription factor NFkappaB in the neutrophils has been associated with the pathogenesis of sepsis, acute lung injury (ALI), bronchopulmonary dysplasia (BPD), and other neutrophil-mediated inflammatory disorders. Despite recent progress in analyzing early NFkappaB activation in human neutrophils, activation of NFkappaB in persistently stimulated neutrophils has not been previously studied. Because it is the persistent NFkappaB activation that is thought to be involved in the host response to sepsis and the pathogenesis of ALI and BPD, we hypothesized that continuously stimulated human neutrophils may exhibit a late phase of NFkappaB activity. The goal of this study was to analyze the NFkappaB activation and expression of IkappaB and NFkappaB proteins during neutrophil stimulation with inflammatory signals for prolonged times. We demonstrate that neutrophil stimulation with lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNFalpha) induces, in addition to the early activation at 30-60 min, a previously unrecognized late phase of NFkappaB activation. In LPS-stimulated neutrophils, this NFkappaB activity typically had a biphasic character, whereas TNFalpha-stimulated neutrophils exhibited a continuous NFkappaB activity peaking around 9 h after stimulation. In contrast to the early NFkappaB activation that inversely correlates to the nuclear levels of IkappaBalpha, however, in continuously stimulated neutrophils, NFkappaB is persistently activated despite considerable levels of IkappaBalpha present in the nucleus. Our data suggest that NFkappaB is persistently activated in human neutrophils during neutrophil-mediated inflammatory disorders, and this persistent NFkappaB activity may represent one of the underlying mechanisms for the continuous production of proinflammatory mediators.

Prolonged exposure to hyperoxia induces pulmonary epithelial cell death and acute lung injury. Although both apoptotic and nonapoptotic morphologies are observed in hyperoxic animal lungs, nonapoptotic cell death had only been recorded in transformed lung epithelium cultured in hyperoxia. To test whether the nonapoptotic characteristics in hyperoxic animal lungs are direct effects of hyperoxia, the mode of cell death was determined both morphologically and biochemically in human primary lung epithelium exposed to 95% O(2). In contrast to characteristics observed in apoptotic cells, hyperoxia induced swelling of nuclei and an increase in cell size, with no evidence for any augmentation in the levels of either caspase-3 activity or annexin V incorporation. These data suggest that hyperoxia can directly induce nonapoptotic cell death in primary lung epithelium. Although hyperoxia-induced nonapoptotic cell death was associated with NF-kappaB activation, it is unknown whether NF-kappaB activation plays any causal role in nonapoptotic cell death. This study shows that inhibition of NF-kappaB activation can accelerate hyperoxia-induced epithelial cell death in both primary and transformed lung epithelium. Corresponding to the reduced cell survival in hyperoxia, the levels of MnSOD were also low in NF-kappaB-deficient cells. These results demonstrate that NF-kappaB protects lung epithelial cells from hyperoxia-induced nonapoptotic cell death.

An increase in polymorphonuclear leukocytes (PMNs) and proinflammatory chemokines, such as IL-8 and macrophage inflammatory protein-1 alpha (MIP), are found in the airways during early stages of bronchopulmonary dysplasia. We determined whether IL-10 produces a dose-related inhibition of proinflammatory chemokine release from stimulated neutrophils of the newborn and whether the mechanism involves the pivotal transcription factor, nuclear factor-kappa B. PMNs isolated from the cord blood of healthy newborns were stimulated submaximally with either lipopolysaccharide (n = 5) or tumor necrosis factor (n = 4), with and without IL-10 (0.01-1000 ng/mL). IL-8 and MIP release were measured in cell culture supernatants at 18 h. The presence or absence of nuclear factor-kappa B activity and inhibitor-kappa B alpha degradation was measured at 30 min and 3 h after PMN stimulation began. During lipopolysaccharide stimulation, IL-10 significantly reduced IL-8 levels from 50 +/- 16 ng/mL to 7 +/- 3 ng/mL, and MIP levels from 14 +/- 5 to 0.7 +/- 0.1 ng/mL (mean +/- SEM, p < 0.01). IL-10 produced an insignificant reduction in IL-8 and MIP levels after stimulation of PMNs with tumor necrosis factor. IL-10 did not inhibit nuclear factor-kappa B activation and inhibitor-kappa B alpha degradation in PMNs stimulated with tumor necrosis factor or lipopolysaccharide for 30 min. After PMN stimulation for 3 h, inhibitor-kappa B alpha cytoplasmic levels were restored; however, they were unaffected by IL-10. We conclude that IL-10 is a potent inhibitor of lipopolysaccharide-stimulated release of IL-8 and MIP from neutrophils of the newborn via a mechanism not involving nuclear factor-kappa B activity. Further work is needed to determine whether exogenous IL-10 may be useful for suppressing inflammation in bronchopulmonary dysplasia.

Human neutrophils differ from other cells by containing high amount of IkappaBalpha in the nucleus, and this increased nuclear IkappaBalpha accumulation is associated with the inhibition of NFkappaB activity and increased apoptosis. However, the mechanisms regulating NFkappaB activation and IkappaBalpha degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFkappaB activity and IkappaBalpha degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-delta (PKCdelta) and IkappaB kinase (IKK) inhibit the OA-induced activation of NFkappaB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFkappaB is regulated by a continuous phosphorylation and degradation of the nuclear IkappaBalpha.

Neutrophils are among the first circulating leukocytes involved in acute inflammatory processes. Transcription factor NF-kappaB plays a key role in the inflammatory response, regulating the expression of proinflammatory and anti-apoptotic genes. Recently we have shown that human neutrophils contain a significant amount of NF-kappaB inhibitor, IkappaBalpha, in the nucleus of unstimulated cells. The present objective was to examine the mechanisms controlling the nuclear content of IkappaBalpha in human neutrophils and to determine whether increased accumulation of IkappaBalpha in the nucleus is associated with increased neutrophil apoptosis. We show for the first time that neutrophil stimulation with pro-inflammatory signals results in degradation of IkappaBalpha that occurs in both cytoplasm and nucleus. Prolonged (2-h) stimulation with TNF and LPS induces resynthesis of IkappaBalpha that is again translocated to the nucleus in human neutrophils, but not in monocytic cells. Leptomycin B, a specific inhibitor of nuclear export, increases nuclear accumulation of IkappaBalpha in stimulated neutrophils by blocking the IkappaBalpha nuclear export, and this is associated with inhibition of NF-kappaB activity, induction of caspase-3 activation, and apoptosis. Based on our data we present a new model of NF-kappaB regulation in human neutrophils by nuclear IkappaBalpha. Our results demonstrate that the NF-kappaB activity in human neutrophils is regulated by mechanisms clearly different from those in monocytes and other human cells and suggest that the increased nuclear content of IkappaBalpha in human neutrophils might represent one of the underlying mechanisms for the increased apoptosis in these cells.

We have observed elevated NF-kappaB DNA-binding activity in nuclear extracts from human papillomavirus type 6- and 11-infected laryngeal papilloma tissues. The predominant DNA-binding species is the p50/p50 homodimer. The elevated NF-kappaB activity could be correlated with a reduced level of cytoplasmic IkappaBbeta and could be associated with the overexpression of p21(CIP1/WAF1) in papilloma cells. Increased NF-kappaB activity and cytoplasmic accumulation of p21(CIP1/WAF1) might counteract death-promoting effects elicited by overexpressed PTEN and reduced activation of Akt and STAT3 previously noted in these tissues.

Expression of the proinflammatory and proangiogenic chemokine IL-8, which is regulated at the transcriptional level by NF-κB, is constitutively increased in androgen-independent metastatic prostate cancer and correlates with poor prognosis. Inhibition of NF-κB–dependent transcription was used as an anticancer strategy for the development of the first clinically approved 26S proteasome inhibitor, bortezomib (BZ). Even though BZ has shown remarkable antitumor activity in hematological malignancies, it has been less effective in prostate cancer and other solid tumors; however, the mechanisms have not been fully understood. In this article, we report that proteasome inhibition by BZ unexpectedly increases IL-8 expression in androgen-independent prostate cancer PC3 and DU145 cells, whereas expression of other NF-κB–regulated genes is inhibited or unchanged. The BZ-increased IL-8 expression is associated with increased in vitro p65 NF-κB DNA binding activity and p65 recruitment to the endogenous IL-8 promoter. In addition, proteasome inhibition induces a nuclear accumulation of IκB kinase (IKK)α, and inhibition of IKKα enzymatic activity significantly attenuates the BZ-induced p65 recruitment to IL-8 promoter and IL-8 expression, demonstrating that the induced IL-8 expression is mediated, at least partly, by IKKα. Together, these data provide the first evidence, to our knowledge, for the gene-specific increase of IL-8 expression by proteasome inhibition in prostate cancer cells and suggest that targeting both IKKα and the proteasome may increase BZ effectiveness in treatment of androgen-independent prostate cancer. [ABSTRACT FROM AUTHOR]