Claus Scheidereit

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

The pathogenesis and etiology of Hodgkins 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 Hodgkins lymphoma cells from undergoing apoptosis under stress conditions. Consistent with this dual function, Hodgkins 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 Hodgkins disease and for developing new therapeutic strategies against it.

NF-κB Function in Growth Control: Regulation of Cyclin D1 Expression and G0/G1-to-S-Phase Transition

ABSTRACT Nuclear factor kappa B (NF-κB) has been implicated in the regulation of cell proliferation, transformation, and tumor development. We provide evidence for a direct link between NF-κB activity and cell cycle regulation. NF-κB was found to stimulate transcription of cyclin D1, a key regulator of G1checkpoint control. Two NF-κB binding sites in the human cyclin D1 promoter conferred activation by NF-κB as well as by growth factors. Both levels and kinetics of cyclin D1 expression during G1phase were controlled by NF-κB. Moreover, inhibition of NF-κB caused a pronounced reduction of serum-induced cyclin D1-associated kinase activity and resulted in delayed phosphorylation of the retinoblastoma protein. Furthermore, NF-κB 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-κB superrepressor (IκBαΔN) could be rescued by ectopic expression of cyclin D1. Thus, NF-κB contributes to cell cycle progression, and one of its targets might be cyclin D1.

IκB kinase complexes: gateways to NF-κB activation and transcription

Transcription factors of the NF-κB family regulate hundreds of genes in the context of multiple important physiological and pathological processes. NF-κB activation depends on phosphorylation-induced proteolysis of inhibitory IκB molecules and NF-κB precursors by the ubiquitin-proteasome system. Most of the diverse signaling pathways that activate NF-κB converge on IκB kinases (IKK), which are essential for signal transmission. Many important details of the composition, regulation and biological function of IKK have been revealed in the last years. This review summarizes current aspects of structure and function of the regular stoichiometric components, the regulatory transient protein interactions of IKK and the mechanisms that contribute to its activation, deactivation and homeostasis. Both phosphorylation and ubiquitinatin (destructive as well as non-destructive) are crucial post-translational events in these processes. In addition to controlling induced IκB degradation in the cytoplasm and processing of the NF-κB precursor p100, nuclear IKK components have been found to act directly at the chromatin level of induced genes and to mediate responses to DNA damage. Finally, IKK is engaged in cross talk with other pathways and confers functions independently of NF-κB.

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.

Aberrantly expressed c‐Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF‐κB

AP‐1 family transcription factors have been implicated in the control of proliferation, apoptosis and malignant transformation. However, their role in oncogenesis is unclear and no recurrent alterations of AP‐1 activities have been described in human cancers. Here, we show that constitutively activated AP‐1 with robust c‐Jun and JunB overexpression is found in all tumor cells of patients with classical Hodgkins disease. A similar AP‐1 activation is present in anaplastic large cell lymphoma (ALCL), but is absent in other lymphoma types. Whereas c‐Jun is up‐regulated by an autoregulatory process, JunB is under control of NF‐κB. Activated AP‐1 supports proliferation of Hodgkin cells, while it suppresses apoptosis of ALCL cells. Furthermore, AP‐1 cooperates with NF‐κB and stimulates expression of the cell‐cycle regulator cyclin D2, proto‐oncogene c‐met and the lymphocyte homing receptor CCR7, which are all strongly expressed in primary HRS cells. Together, these data suggest an important role of AP‐1 in lymphoma pathogenesis.

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

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

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

The proto-oncoprotein Bcl-3 is a member of the IκB 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-κB 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 Polll holoenzyme component Brca1, respectively. Bcl-3, p50, and either Bard1, Tip60 or Pirin are sequestered into quarternary complexes on NF-κB 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-κB binding site, indicating that quarternary complexes containing Bcl-3 interactors modulate NF-κB driven gene expression. These data implicate Bcl-3 as an adaptor between NF-κB 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.

The NF-κB/Rel and IκB gene families: mediators of immune response and inflammation

Abstract Two families of gene regulators play an important role in cellular signaling processes in vertebrates: the nuclear factor κB (NF-κB)/Rel group of transcription activators and their coevolved regulatory proteins, the inhibitors of κB (IκBs). The biological functions of NF-κB 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-κB system are deregulated and could thus present potential diagnostic probes or targets for therapeutic intervention.

Nuclear Factor κB–dependent Gene Expression Profiling of Hodgkin's Disease Tumor Cells, Pathogenetic Significance, and Link to Constitutive Signal Transducer and Activator of Transcription 5a Activity

Constitutive nuclear nuclear factor (NF)-κB activity is observed in a variety of hematopoietic and solid tumors. Given the distinctive role of constitutive NF-κB for Hodgkin and Reed-Sternberg (HRS) cell viability, we performed molecular profiling in two Hodgkins disease (HD) cell lines to identify NF-κB target genes. We recognized 45 genes whose expression in both cell lines was regulated by NF-κB. The NF-κB–dependent gene profile comprises chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors, the majority of which maintain a marker-like expression in HRS cells. Remarkably, we found 17 novel NF-κB target genes. Using chromatin immunoprecipitation we demonstrate that NF-κB is recruited directly to the promoters of several target genes, including signal transducer and activator of transcription (STAT)5a, interleukin-13, and CC chemokine receptor 7. Intriguingly, NF-κB positively regulates STAT5a expression and signaling pathways in HRS cells, and promotes its persistent activation. In fact, STAT5a overexpression was found in most tumor cells of tested patients with classical HD, indicating a critical role for HD. The gene profile underscores a central role of NF-κB in the pathogenesis of HD and potentially of other tumors with constitutive NF-κB activation.

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

The pleiotropic transcription factor NF-κB controls cellular apoptotic and growth processes and increasing evidence suggests a role in tumorigenesis. We describe here that constitutively activated NF-κB 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 IκBα, indicating that activation of upstream kinases, which trigger IκBα degradation, has led to nuclear translocation of NF-κB. 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 α-1 antitrypsin. Constitutive NF-κB activation is thus a common characteristic of childhood ALL and strongly suggests a critical role of this factor for leukemia cell survival.