Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells.

The pathogenesis and etiology of Hodgkin's disease, a common human malignant lymphoma, is still unresolved. As a unique characteristic, we have identified constitutive activation of the transcription factor nuclear factor (NF)-kappaB p50-RelA in Hodgkin/Reed-Sternberg (H/RS) cells, which discriminates these neoplastic cells from most cell types studied to date. In contrast to other lymphoid and nonlymphoid cell lines tested, proliferation of H/RS cells depended on activated NF-kappaB. Furthermore, constitutive NF-kappaB p50-RelA prevented Hodgkin's lymphoma cells from undergoing apoptosis under stress conditions. Consistent with this dual function, Hodgkin's lymphoma cells depleted of constitutive nuclear NF-kappaB revealed strongly impaired tumor growth in severe combined immunodeficient mice. Our findings identify NF-kappaB as an important component for understanding the pathogenesis of Hodgkin's disease and for developing new therapeutic strategies against it.

NF-kappaB and the innate immune response.

In the innate immune reaction, microbial pathogens activate phylogenetically conserved cellular signal transduction pathways that regulate the ubiquitous nuclear factor-kappaB (NFkappaB). NF-kappaB has pleiotropic functions in immunity; however, it is also critical for development and cellular survival. Many aspects of how the different pathways utilize a common kinase complex that ultimately activates NF-kappaB have been clarified by gene inactivation and biochemical analysis.

Shared pathways of IkappaB kinase-induced SCF(betaTrCP)-mediated ubiquitination and degradation for the NF-kappaB precursor p105 and IkappaBalpha.

p105 (NFKB1) acts in a dual way as a cytoplasmic IkappaB molecule and as the source of the NF-kappaB p50 subunit upon processing. p105 can form various heterodimers with other NF-kappaB subunits, including its own processing product, p50, and these complexes are signal responsive. Signaling through the IkappaB kinase (IKK) complex invokes p105 degradation and p50 homodimer formation, involving p105 phosphorylation at a C-terminal destruction box. We show here that IKKbeta phosphorylation of p105 is direct and does not require kinases downstream of IKK. p105 contains an IKK docking site located in a death domain, which is separate from the substrate site. The substrate residues were identified as serines 923 and 927, the latter of which was previously assumed to be a threonine. S927 is part of a conserved DSGPsi motif and is functionally most critical. The region containing both serines is homologous to the N-terminal destruction box of IkappaBalpha, -beta, and -epsilon. Upon phosphorylation by IKK, p105 attracts the SCF E3 ubiquitin ligase substrate recognition molecules betaTrCP1 and betaTrCP2, resulting in polyubiquitination and complete degradation by the proteasome. However, processing of p105 is independent of IKK signaling. In line with this and as a physiologically relevant model, lipopolysaccharide (LPS) induced degradation of endogenous p105 and p50 homodimer formation, but not processing in pre-B cells. In mutant pre-B cells lacking IKKgamma, processing was unaffected, but LPS-induced p105 degradation was abolished. Thus, a functional endogenous IKK complex is required for signal-induced p105 degradation but not for processing.

B-cell receptor- and phorbol ester-induced NF-kappaB and c-Jun N-terminal kinase activation in B cells requires novel protein kinase C's.

Antigen receptor signaling is known to activate NF-kappaB in lymphocytes. While T-cell-receptor-induced NF-kappaB activation critically depends on novel protein kinase C theta (PKCtheta), the role of novel PKCs in B-cell stimulation has not been elucidated. In primary murine splenic B cells, we found high expression of the novel PKCs delta and epsilon but only weak expression of the theta isoform. Rottlerin blocks phorbol ester (phorbol myristate acetate [PMA])- or B-cell receptor (BCR)-mediated NF-kappaB and c-Jun N-terminal kinase (JNK) activation in primary B and T cells to a similar extent, suggesting that novel PKCs are positive regulators of signaling in hematopoietic cells. Mouse 70Z/3 pre-B cells have been widely used as a model for NF-kappaB activation in B cells. Similar to the situation in splenic B cells, rottlerin inhibits BCR and PMA stimulation of NF-kappaB in 70Z/3 cells. A derivative of 70Z/3 cells, 1.3E2 cells, are defective in NF-kappaB activation due to the lack of the IkappaB kinase (IKKgamma) protein. Ectopic expression of IKKgamma can rescue NF-kappaB activation in response to lipopolysaccharides (LPS) and interleukin-1beta (IL-1beta), but not to PMA. In addition, PMA-induced activation of the mitogen-activated protein kinase JNK is blocked in 1.3E2 cells, suggesting that an upstream component common to both pathways is either missing or mutated. Analysis of various PKC isoforms revealed that exclusively PKCtheta was absent in 1.3E2 cells while it was expressed in 70Z/3 cells. Stable expression of either novel PKCtheta or -delta but not classical PKCbetaII in 1.3E2 IKKgamma-expressing cells rescues PMA activation of NF-kappaB and JNK signaling, demonstrating a critical role of novel PKCs for B-cell activation.

Maintenance of NF-kappa B activity is dependent on protein synthesis and the continuous presence of external stimuli.

The activation of NF-kappa B-like activities (called NF-kappa B) by tumor necrosis factor alpha (TNF alpha) and the phorbol ester phorbol 12-myristate 13-acetate (PMA) were compared. High levels of NF-kappa B activity were found 2 to 4 min after TNF alpha addition to human HL60 cells and lasted for at least 3 h, although the half-life of active NF-kappa B was less than 30 min. Inactive NF-kappa B, however, was relatively stable. NF-kappa B activation by TNF alpha was initially cycloheximide insensitive, but maintenance of NF-kappa B activity required ongoing protein synthesis and continuous stimulation by TNF alpha. Thus, the cells did not remain in an activated state without stimulation. In HL60 cells, NF-kappa B induction by PMA required 30 to 45 min and was completely dependent on de novo protein synthesis, while PMA (and interleukin-1) induced NF-kappa B activity rapidly in mouse 70Z/3 cells via a protein synthesis-independent mechanism. The NF-kappa B-like activities obtained under each condition behaved identically in methylation interference and native proteolytic fingerprinting assays. The NF-kappa B-like factors induced are thus all very similar or identical. We suggest that cell-specific differences in the protein kinase C-dependent activation of NF-kappa B may exist and that TNF alpha and PMA may induce expression of the gene(s) encoding NF-kappa B.

NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition.

Nuclear factor kappa B (NF-kappaB) has been implicated in the regulation of cell proliferation, transformation, and tumor development. We provide evidence for a direct link between NF-kappaB activity and cell cycle regulation. NF-kappaB was found to stimulate transcription of cyclin D1, a key regulator of G1 checkpoint control. Two NF-kappaB binding sites in the human cyclin D1 promoter conferred activation by NF-kappaB as well as by growth factors. Both levels and kinetics of cyclin D1 expression during G1 phase were controlled by NF-kappaB. Moreover, inhibition of NF-kappaB caused a pronounced reduction of serum-induced cyclin D1-associated kinase activity and resulted in delayed phosphorylation of the retinoblastoma protein. Furthermore, NF-kappaB promotes G1-to-S-phase transition in mouse embryonal fibroblasts and in T47D mammary carcinoma cells. Impaired cell cycle progression of T47D cells expressing an NF-kappaB superrepressor (IkappaBalphaDeltaN) could be rescued by ectopic expression of cyclin D1. Thus, NF-kappaB contributes to cell cycle progression, and one of its targets might be cyclin D1.

Alternative splicing variants of IkappaB beta establish differential NF-kappaB signal responsiveness in human cells.

To release transcription factor NF-kappaB into the nucleus, the mammalian IkappaB molecules IkappaB alpha and IkappaB beta are inactivated by phosphorylation and proteolytic degradation. Both proteins contain conserved signal-responsive phosphorylation sites and have conserved ankyrin repeats. To confer specific physiological functions to members of the NF-kappaB/Rel family, the different IkappaB molecules could vary in their specific NF-kappaB/Rel factor binding activities and could respond differently to activation signals. We have demonstrated that both mechanisms apply to differential regulation of NF-kappaB function by IkappaB beta relative to IkappaB alpha. Via alternative RNA processing, human IkappaB beta gives rise to different protein isoforms. IkappaB beta1 and IkappaB beta2, the major forms in human cells, differ in their carboxy-terminal PEST sequences. IkappaB beta2 is the most abundant species in a number of human cell lines tested, whereas IkappaB beta1 is the only form detected in murine cells. These isoforms are indistinguishable in their binding preferences to cellular NF-kappaB/Rel homo- and heterodimers, which are distinct from those of IkappaB alpha, and both are constitutively phosphorylated. In unstimulated B cells, however, IkappaB beta1, but not IkappaB beta2, is found in the nucleus. Furthermore, the two forms differ markedly in their efficiency of proteolytic degradation after stimulation with several inducing agents tested. While IkappaB beta1 is nearly as responsive as IkappaB alpha, indicative of a shared activation mechanism, IkappaB beta2 is only weakly degraded and often not responsive at all. Alternative splicing of the IkappaB beta pre-mRNA may thus provide a means to selectively control the amount of IkappaB beta-bound NF-kappaB heteromers to be released under NF-kappaB stimulating conditions.

Functional interference of Sp1 and NF-kappaB through the same DNA binding site.

Gene activation by NF-kappaB/Rel transcription factors is modulated by synergistic or antagonistic interactions with other promoter-bound transcription factors. For example, Sp1 sites are often found in NF-kappaB-regulated genes, and Sp1 can activate certain promoters in synergism with NF-kappaB through nonoverlapping binding sites. Here we report that Sp1 acts directly through a subset of NF-kappaB binding sites. The DNA binding affinity of Sp1 to these NF-kappaB sites, as determined by their relative dissociation constants and their relative efficiencies as competitor DNAs or as binding site probes, is in the order of that for a consensus GC box Sp1 site. In contrast, NF-kappaB does not bind to a GC box Sp1 site. Sp1 can activate transcription through immunoglobulin kappa-chain enhancer or P-selectin promoter NF-kappaB sites. p50 homodimers replace Sp1 from the P-selectin promoter by binding site competition and thereby either inhibit basal Sp1-driven expression or, in concert with Bcl-3, stimulate expression. The interaction of Sp1 with NF-kappaB sites thus provides a means to keep an elevated basal expression of NF-kappaB-dependent genes in the absence of activated nuclear NF-kappaB/Rel.

Mixed-lineage kinase 3 delivers CD3/CD28-derived signals into the IkappaB kinase complex.

The phosphorylation of IkappaB by the multiprotein IkappaB kinase complex (IKC) precedes the activation of transcription factor NF-kappaB, a key regulator of the inflammatory response. Here we identified the mixed-lineage group kinase 3 (MLK3) as an activator of NF-kappaB. Expression of the wild-type form of this mitogen-activated protein kinase kinase kinase (MAPKKK) induced nuclear immigration, DNA binding, and transcriptional activity of NF-kappaB. MLK3 directly phosphorylated and thus activated IkappaB kinase alpha (IKKalpha) and IKKbeta, revealing its function as an IkappaB kinase kinase (IKKK). MLK3 cooperated with the other two IKKKs, MEKK1 and NF-kappaB-inducing kinase, in the induction of IKK activity. MLK3 bound to components of the IKC in vivo. This protein-protein interaction was dependent on the central leucine zipper region of MLK3. A kinase-deficient version of MLK3 strongly impaired NF-kappaB-dependent transcription induced by T-cell costimulation but not in response to tumor necrosis factor alpha or interleukin-1. Accordingly, endogenous MLK3 was phosphorylated and activated by T-cell costimulation but not by treatment of cells with tumor necrosis factor alpha or interleukin-1. A dominant negative version of MLK3 inhibited NF-kappaB- and CD28RE/AP-dependent transcription elicited by the Rho family GTPases Rac and Cdc42, thereby providing a novel link between these GTPases and the IKC.

NF-kappa B precursor p100 inhibits nuclear translocation and DNA binding of NF-kappa B/rel-factors.

The NF-kappa B precursor p100 (lyt-10, p97, p98) generates after proteolytic processing a 52 kDa subunit, which can bind to kappa B-motifs. A deregulated form of the p100 gene, which is structurally altered by a t(10;14) translocation, has a potential oncogenic role in certain human B cell lymphomas. In this study p100 was analysed for its ability to interact with its own processing product p52, with p50, the product of the NF-kappa B precursor p105, and with other NF-kappa B/rel-proteins. As demonstrated by a combination of Western blot analysis, band shift analysis and indirect immunofluorescence labelling of transfected cells, p100 itself was localized in the cytoplasm and indiscriminately retained each co-expressed NF-kappa B subunit. Thereby it simultaneously inhibited their DNA binding activities. Thus, a major function of p100 is, like p105, to associate with subunits of the rel multigene family in the cytoplasm in an I kappa B-like fashion. The similarity between p100 and p105 is also reflected by equivalent protein interactions of their processing products: like NF-kappa B-p50, also NF-kappa B-p52 heteromerised promiscuously with all rel-factors tested. Moreover, p52 efficiently interacts with the candidate oncogene product Bcl-3 and also binds to the basic-leucine zipper protein NF-IL6.

Molecular mechanisms of constitutive NF-kappaB/Rel activation in Hodgkin/Reed-Sternberg cells.

A common characteristic of malignant cells derived from patients with Hodgkin's disease (HD) is a high level of constitutive nuclear NF-kappaB/Rel activity, which stimulates proliferation and confers resistance to apoptosis. We have analysed the mechanisms that account for NF-kappaB activation in a panel of Hodgkin/Reed-Sternberg (H-RS) cell lines. Whereas two cell lines (L428 and KMH-2) expressed inactive IkappaBalpha, no significant changes in NF-kappaB or IkappaB expression were seen in other H-RS cells (L591, L1236 and HDLM-2). Constitutive NF-kappaB was susceptible to inhibition by recombinant IkappaBalpha, suggesting that neither mutations in the NF-kappaB genes nor posttranslational modifications of NF-kappaB were involved. Endogenous IkappaBalpha was bound to p65 and displayed a very short half-life. IkappaBalpha degradation could be blocked by inhibitors of the NF-kappaB activating pathway. Proteasomal inhibition caused an accumulation of phosphorylated IkappaBalpha and a reduction of NF-kappaB activity in HDLM-2 and L1236 cells. By in vitro kinase assays we demonstrate constitutive IkappaB kinase (IKK) activity in H-RS cells, indicating ongoing signal transduction. Furthermore, H-RS cells secrete one or more factor(s) that were able to trigger NF-kappaB activation. We conclude that aberrant activation of IKK's, and in some cases defective IkappaBs, lead to constitutive nuclear NF-kappaB activity, which in turn results in a growth advantage of Hodgkin's disease tumor cells.

The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators.

The proto-oncoprotein Bcl-3 is a member of the IkappaB family and is present predominantly in the nucleus. To gain insight into specific nuclear functions of Bcl-3 we have isolated proteins that interact with its ankyrin repeat domain. Using the yeast two-hybrid-system we identified four novel binding partners of Bcl-3 in addition to NF-kappaB p50 and p52, previously known to associate with Bcl-3. The novel Bcl-3 interactors Jab1, Pirin, Tip60 and Bard1 are nuclear proteins which also bind to other transcription factors including c-Jun, nuclear factor I (NFI), HIV-1 Tat or the tumor suppressor and PolII holoenzyme component Brca1, respectively. Bcl-3, p50, and either Bard1, Tip60 or Pirin are sequestered into quarternary complexes on NF-kappaB DNA binding sites, whereas Jab1 enhances p50-Bcl-3-DNA complex formation. Furthermore, the histone acetylase Tip60 enhances Bcl-3-p50 activated transcription through an NF-kappaB binding site, indicating that quarternary complexes containing Bcl-3 interactors modulate NF-kappaB driven gene expression. These data implicate Bcl-3 as an adaptor between NF-kappaB p50/p52 and other transcription regulators and suggest that its gene activation function may at least in part be due to recruitment of the Tip60 histone actetylase.

Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-kappaB and induces tissue factor and PAI-1 expression: a potential link to accelerated arteriosclerosis.

BACKGROUND: Recent reports link C. pneumoniae infection of arteriosclerotic lesions to the precipitation of acute coronary syndromes, which also feature tissue factor and plasminogen activator inhibitor 1 (PAI-1) overexpression. We investigated whether or not C. pneumoniae can induce thrombogenicity by upregulation of procoagulant proteins. METHODS AND RESULTS: Human vascular endothelial and smooth muscle cells were infected with a strain of C. pneumoniae isolated from an arteriosclerotic coronary artery. Tissue factor, PAI-1, and interleukin-6 expression was increased in infected cells. Concomitantly, NF-kappaB was activated and IkappaBalpha degraded. p50/p65 heterodimers were identified as the components responsible for the NF-kappaB activity. CONCLUSIONS: These data provide evidence that C. pneumoniae infection can induce procoagulant protein and proinflammatory cytokine expression. This cellular response is accompanied by activation of NF-kappaB. Our results demonstrate how C. pneumoniae infection may initiate acute coronary syndromes.

Transcription factor NF-kappaB is constitutively activated in acute lymphoblastic leukemia cells.

The pleiotropic transcription factor NF-kappaB controls cellular apoptotic and growth processes and increasing evidence suggests a role in tumorigenesis. We describe here that constitutively activated NF-kappaB complexes are found in the vast majority (39 out of 42 samples) of childhood acute lymphoblastic leukemia (ALL) without any subtype restriction. Electrophoretic shift analysis further demonstrates that these complexes are composed of p50-p50 and p65-p50 dimers. Proteasome inhibition in primary ALL cultures results in a hyperphosphorylated form of IkappaBalpha, indicating that activation of upstream kinases, which trigger IkappaBalpha degradation, has led to nuclear translocation of NF-kappaB. Careful inhibition of cellular proteolytic activities is of importance when analyzing extracts from primary ALL cells. Degradation of p65 and other proteins in ALL samples could be specifically suppressed by alpha-1 antitrypsin. Constitutive NF-kappaB activation is thus a common characteristic of childhood ALL and strongly suggests a critical role of this factor for leukemia cell survival.

In vitro susceptibility to TRAIL-induced apoptosis of acute leukemia cells in the context of TRAIL receptor gene expression and constitutive NF-kappa B activity.

The TNF-related apoptosis-inducing ligand (TRAIL) is currently under evaluation as a possible (co-)therapeutic in cancer treatment. We therefore examined 129 cell samples from patients with de novo acute leukemia as to their constitutive susceptibility to TRAIL-induced apoptosis In vitro. Only 21 (16%) cell samples revealed at least 10% TRAIL-susceptible cells/sample as detected by flow cytometric annexinV staining after 24 h culture compared with medium control. Precursor B cell ALL samples (11 (27%) of 41) were more TRAIL-susceptible compared with AML (5 (9%) of 54; P < 0.05) but not compared with precursor T cell ALL (5 (15%) of 34; P = 0.20). Furthermore, we examined constitutive mRNA expression levels of TRAIL receptors R1-R4 by semi-quantitative RT-PCR (n = 58). Expression levels were heterogeneous, however, there was no significant correlation between the expression of the signal-transducing receptors (R1, R2) as well as of the decoy receptors (R3, R4) and TRAIL sensitivity in this series. Constitutive NF-kappa B activity has been shown to influence TRAIL susceptibility of leukemic cells. In 39 leukemic cell samples examined, we found a generally high NF-kappa B activity as detected by electrophoretic mobility shift assay which did not differ between TRAIL-susceptible and TRAIL-resistant cases. Finally, 49 acute leukemic cell samples were coincubated with doxorubicin in vitro. Doxorubicin sensitized four of 35 initially TRAIL-resistant samples and augmented TRAIL-induced apoptosis in two of 14 TRAIL-susceptible samples. In summary, constitutive TRAIL susceptibility differs between leukemia subtypes and does not correlate with mRNA expression levels of the TRAIL receptors R1-R4 as well as constitutive NF-kappa B activation status. The observed sensitization of leukemic cells to TRAIL by doxorubicin in vitro indicates that TRAIL should be further evaluated as to its possible role as an in vivo cotherapeutic in acute leukemia.

The NF-kappa B/Rel and I kappa B gene families: mediators of immune response and inflammation.

Two families of gene regulators play an important role in cellular signaling processes in vertebrates: the nuclear factor kappa B (NF-kappa B)/Rel group of transcription activators and their coevolved regulatory proteins, the inhibitors of kappa B (I kappa Bs). The biological functions of NF-kappa B comprise communication between cells, embryonal development, the response to stress, inflammation and viral infection, and the maintenance of cell type specific expression of genes. In several pathogenic conditions components of the NF-kappa B system are deregulated and could thus present potential diagnostic probes or targets for therapeutic intervention.

Regulation of NF-kappa B activity by I kappa B alpha and I kappa B beta stability.

Transcription factor NF-kappa B must be released from cytoplasmic inhibitory molecules (I kappa Bs) in order to move to the nucleus and to activate its target genes. Little is known about the mechanisms regulating the maintenance of constitutive nuclear NF-kappa B in some cell-types and of sustained nuclear NF-kappa B activity after stimulation. Increased turnover has been implicated in the regulation of constitutive NF-kappa B activity in mature B cells. We therefore compared the turnover of I kappa B alpha and I kappa B beta in mature B cells and HeLa cells. Both proteins display a high turnover in B cells although I kappa B beta is considerably more stable than I kappa B alpha. The half-life of both inhibitors is increased in HeLa cells. In contrast, all other NF-kappa B/I kappa B molecules tested are relatively stable in both cell-types. The elevated turnover of endogenous I kappa B alpha in Namalwa cells is inhibited by a proteasome inhibitor and thus seems to be driven by the same degradation machinery as the slower turnover in non-B cells. Furthermore, we investigated the processes involved in persistent activation of NF-kappa B. TNF-alpha signaling leads to a rapid depletion of cellular I kappa B beta pools. I kappa B alpha is efficiently resynthesized whereas I kappa B beta levels stay low for a prolonged time. NF-kappa B binding activity can be detected for several hours after stimulation. We found that removal of the TNF-alpha containing medium causes a rapid decrease in nuclear NF-kappa B. A phosphoform of newly synthesized I kappa B alpha is visible when degradation by the proteasome is inhibited and new I kappa B alpha displays the same properties regarding phosphorylation and degradation in response to a second inducer. There is no significant difference in the turnover of pre- and post-inductive I kappa B alpha. These observations suggest that resynthesis of I kappa B alpha and removal of the stimulus are obligatory steps for the inactivation of nuclear NF kappa B.

Neither the endogenous nor a functional steroid hormone receptor binding site transactivate the ribosomal RNA gene promoter in vitro.

The mammalian ribosomal RNA gene promoters exhibit a conserved sequence between positions +1 and +16 that shows a high degree of homology to the response element for glucocorticoids and progestins (GRE/PRE). These sequences bind specifically the glucocorticoid receptor and the progesterone receptor (PR) albeit with lower affinity than a canonical GRE/PRE. Because steroid hormones are known to affect expression of the ribosomal genes, we tested the influence of hormone receptors on the activity of the ribosomal RNA gene promoter in a cell-free transcription assay. Preparations of PR that induce transcription from the mouse mammary tumour virus (MMTV) promoter do not stimulate but slightly inhibit transcription from the ribosomal RNA gene promoter. This weak negative effect is not mediated through binding to the hypothetical GRE/PRE as a mutant promoter that does not bind receptor is equally repressed. Introduction of the functional MMTV GRE/PRE upstream of the basal ribosomal RNA gene promoter does not enhance its transcription in the presence of an active PR. Thus, RNA polymerase I transcription cannot be stimulated in vitro by cis elements and regulatory proteins that are active in RNA polymerase II transcription.

High expression of RelA/p65 is associated with activation of nuclear factor-kappaB-dependent signaling in pancreatic cancer and marks a patient population with poor prognosis.

Activation of nuclear factor-kappaB (NF-kappaB) signaling was observed in pancreatic adenocarcinoma cell lines and tumours. However, information on the expression of RelA/p65, the major transcription activating NF-kappaB subunit, in these carcinomas and possible correlations thereof with NF-kappaB activation and patient survival is not available. To provide this missing translational link, we analysed expression of RelA/p65 in 82 pancreatic adenocarcinomas by immunohistochemistry. Moreover, we measured activation of the NF-kappaB pathway in 11 tumours by quantitative PCR for NF-kappaB target genes. We observed strong cytoplasmic or nuclear expression of RelA/p65 in 42 and 37 carcinomas, respectively. High cytoplasmic and nuclear expression of RelA/p65 had negative prognostic impact with 2-year survival rates for patients without cytoplasmic or nuclear RelA/p65 positivity of 41 and 40% and rates for patients with strong cytoplasmic or nuclear RelA/p65 expression of 22 and 20%, respectively. High RelA/p65 expression was correlated to increased expression of NF-kappaB target genes. The observation that high expression of RelA/p65 is correlated to an activation of the NF-kappaB pathway and indicates poor patient survival identifies a patient subgroup that might particularly benefit from NF-kappaB-inhibiting agents in the treatment of pancreatic cancer. Based on our findings, this subgroup could be identified by applying simple immunohistochemical techniques.