Interleukin-6 (IL-6) is a pleiotropic cytokine, which is involved in inflammatory and immune responses, acute phase reactions, and hematopoiesis. In the mouse fibrosarcoma cell line L929, the nuclear factor (NF)-B plays a crucial role in IL-6 gene expression mediated by tumor necrosis factor (TNF). The levels of the activated factor do not, however, correlate with the variations of IL-6 gene transcription; therefore, other factors and/or regulatory mechanisms presumably modulate the levels of IL-6 mRNA production. Upon analysis of various deletion and point-mutated variants of the human IL-6 gene promoter coupled to a reporter gene, we screened for possible cooperating transcription factors. Even the smallest deletion variant, containing almost exclusively a NF-B-responsive sequence preceding the IL-6 minimal promoter, as well as a recombinant construction containing multiple B-motifs, could still be stimulated with TNF. We observed that the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was able to repress TNF-stimulated expression of the IL-6 gene, as well as of a B-dependent reporter gene construct, without affecting the levels of NF-B binding to DNA. Furthermore, we clearly show that, using a nuclear Gal4 "one-hybrid" system, the MAPK inhibitors SB203580 and PD0980589 have a direct repressive effect on the transactivation potential of the p65 B subunit. Therefore, we conclude that, in addition to cytoplasmic activation and DNA binding of NF-B, the p38 and extracellular signalregulated kinase MAPK pathways act as necessary cooperative mechanisms to regulate TNF-induced IL-6 gene expression by modulating the transactivation machinery.

PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

Expression of the pleiotropic cytokine interleukin (IL)-6 can be stimulated by the proinflammatory cytokine tumor necrosis factor (TNF) and the microbial alkaloid staurosporine (STS). In this report, the transcriptional mechanisms were thoroughly investigated. Whereas transcription factors binding to the activator protein-1-, cAMP-responsive element-, and CAAT enhancer-binding protein-responsive sequences are necessary for gene activation by STS, nuclear factor (NF)-B alone is responsible and sufficient for inducibility by TNF, which reveals distinct signaling pathways for both compounds. At the cofactor level, cAMP-responsive element-binding protein-binding protein (CBP) or p300 potentiate basal and induced IL-6 promoter activation via multiple protein-protein interactions with all transcription factors bound to the promoter DNA. However, the strongest promoter activation relies on the p65 NF-B subunit, which specifically engages CBP/p300 for maximal transcriptional stimulation by its histone acetyltransferase activity. Moreover, treatment of chromatinintegrated promoter constructions with the histone deacetylase inhibitor trichostatin A exclusively potentiates TNF-dependent (i.e. NF-B-mediated) gene activation, while basal or STS-stimulated IL-6 promoter activity remains completely unchanged. Similar observations were recorded with other natural NF-B-driven promoters, namely IL-8 and endothelial leukocyte adhesion molecule (ELAM). We conclude that, within an "enhanceosome-like" structure, NF-B is the central mediator of TNF-induced IL-6 gene expression, involving CBP/p300 and requiring histone acetyltransferase activity.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n ¼ 56), mantle cell lymphoma (n ¼ 54), marginal zone lymphoma (n ¼ 41) and follicular lymphoma (n ¼ 109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Glucocorticoids (GCs) are used to combat inflammatory diseases. Their beneficial effect relies mainly on the inhibition of NF-Band͞or AP-1-driven proinflammatory gene expression. Previously, we have shown that GCs repress tumor necrosis factor-induced IL-6 gene expression by an NF-B-dependent nuclear mechanism without changing the DNA-binding capacity of NF-B or the expression levels of the cytoplasmic inhibitor of NF-B (I B-␣). In the present work, we investigate the effect of GC repression on different natural and͞or recombinant NF-B-driven reporter gene constructs in the presence of increasing amounts of various coactivator molecules, such as CREB-binding protein (CBP), p300, and SRC-1. We found that GCs maintain their repressive capacities, irrespective of the amount of cofactor present in the cell. Similar results were obtained for the reciprocal transrepression of a GC receptor (GR) element-driven reporter gene by p65. We demonstrate that neither the expression levels of p65 and CBP nor their physical association are affected by activated GR. Using Gal4 chimeras, we show that repression by GCs is specific for p65-mediated transactivation, ruling out competition for limiting nuclear factors as the major underlying mechanism of gene repression. In addition, the transactivation potential of a point-mutated Gal4-p65 variant with a decreased CBP interaction capability is still repressed by GR. Finally, we present evidence that the specificity of GC repression on p65-driven gene expression is codetermined by the TATA box context. T he glucocorticoid (GC) receptor (GR) is a ligand-dependenttranscription factor belonging to the superfamily of steroid͞ thyroid hormone receptors. These receptors and their cognate hormones control various aspects of metabolic homeostasis, embryonic development, and physiological stress. Activated GRs modulate transcription by either directly binding to GR elements (GREs) in promoters of positively regulated genes or indirectly binding by association with other transcription factors, such as NF-B or AP-1. The latter function is considered to be very important in the battle against inflammatory and immune diseases, in which GCs are used effectively as therapeutic agents (1, 2).The transcription factor NF-B plays a critical role in immune homeostasis, cell growth, and survival. A persistent activation of this factor compromises health and is associated with inflammatory and neoplastic diseases as well as with viral infection. The mammalian NF-B͞Rel family of proteins consists presently of five members, namely, Rel (c-Rel), p65 (Rel A), Rel B, p50 (NFKB1), and p52 (NFKB2). In general, the designation NF-B refers to the most frequently occurring heterodimeric complex between the p50 and p65 subunits. NF-B activation may be induced by different signals, such as viral infection, the proinflammatory cytokines tumor necrosis factor (TNF) and IL-1, phorbol esters, UV irradiation, and bacterial lipopolysaccharides. These signals lead to phosphorylation and degradation of the inhibitor of NF-B (...

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Analysis by electrophoretic mobility shift assays (EMSA) of the different proteins associated with the B sequence of the interleukin-6 (IL-6) promoter (IL6-B) allowed us to detect a specific complex formed with the recombination signal sequence binding protein J (RBP-J). Single-base exchanges within the oligonucleotide sequence defined the critical base pairs involved in the interaction between RBP-J and the IL6-B motif. Binding analysis suggests that the amount of RBP-J protein present in the nucleus is severalfold higher than the total amount of inducible NF-B complexes but that the latter bind DNA with a 10-fold-higher affinity. A reporter gene study was performed to determine the functional implication of this binding; we found that the constitutive occupancy of the IL6-B site by the RBP-J protein was responsible for the low basal levels of IL-6 promoter activity in L929sA fibrosarcoma cells and that RBP-J partially blocked access of NF-B complexes to the IL-6 promoter. We propose that such a mechanism could be involved in the constitutive repression of the IL-6 gene under normal physiological conditions.

The cytokine tumor necrosis factor (TNF) activates diverse signaling molecules resulting in gene expression, differentiation, and/or cell death. Here we report a novel feature induced by TNF, namely translocation of mitochondria from a dispersed distribution to a perinuclear cluster. Mitochondrial translocation correlated with sensitivity to the cell death-inducing activity of TNF and was mediated by the 55-kDa TNF receptor (TNF-R55), but not by Fas, indicating that the signaling pathway requires a TNF-R55-specific but death domainindependent signal. Indeed, using L929 cells that express mutant TNF-R55, we showed that the membraneproximal region of TNF-R55 was essential for signaling to mitochondrial translocation. In the absence of translocation, the cell death response was markedly delayed, pointing to a cooperative effect on cell death. Translocation of mitochondria, although dependent on the microtubules, was not imposed by the latter and was equally induced by TNF-independent immunoinhibition of the motor protein kinesin. Additionally, immunoinhibition with antibody directed against the tail domain of kinesin synergized with TNF-induced cell death. Based on this functional mimicry, we propose that a TNF-R55 membrane-proximal region-dependent signal impedes mitochondria-associated kinesin, resulting in cooperation with the TNF-R55 death domain-induced cytotoxic response and causing the observed clustering of mitochondria.Mitochondria are the energy-providing organelles in eukaryotic cells. However, accumulating evidence shows that these organelles also have an active function in cell death. Disruption of mitochondrial transmembrane potential (⌬⌿ m ) 1 during apoptosis induced by various stimuli in diverse cell types represents an irreversible commitment to cell death, preceding the late characteristics of apoptosis, such as DNA condensation and degradation as well as formation of apoptotic bodies (1, 2). A causative link between ⌬⌿ m and nuclear apoptosis is supported by the release of a caspase-like, apoptosis-inducing factor from the mitochondrial intermembrane space after permeability transition, a condition leading to ⌬⌿ m disruption (3). In addition to apoptosis-inducing factor, cytochrome c induces apoptosis in a cell-free system in the presence of dATP and cytosolic extracts (4). The release of cytochrome c from mitochondria is independent of permeability transition and ⌬⌿ m disruption, suggesting a possible role in apoptosis in cell types that do not exhibit disruption of ⌬⌿ m (5, 6). Moreover, Bcl-2 prevents release of apoptosis-inducing factor and cytochrome c from the mitochondria (3, 5, 6), and Bcl-x L inhibits the accumulation of cytochrome c in the cytosol during apoptosis possibly by binding to it and thus blocking its availability (7). Therefore, the antiapoptotic role of Bcl-2 and family members could be based on counteracting mitochondrial dysfunction and subsequent release of apoptogenic factors. Mitochondrial dysfunction also plays a crucial role in cell types that exhibit necrosis-like c...