No abstract

Pancreatic carcinogenesis is a multistage process that involves numerous genetic alterations, including activating mutations of the Ki-Ras protooncogene by codon 12 mutations. 1 Furthermore, a variety of growth factors and their receptors are found overexpressed and may combine to provide a growth advantage to human pancreatic cancer cells. 2,3 However, downstream targets and in particular nuclear events involved in pancreatic cancer cell growth are poorly understood.Nuclear factor kappa B (NF-B) is a central regulator of the immune response and apoptosis. NF-B/Rel has been implicated in the pathogenesis of certain tumors, especially those of hematopoietic origin. In most cell types, NF-B dimers are sequestered as inactive cytoplasmic complexes by binding to their inhibitors, IBs. A common key event in the activation of NF-B is the inducible phosphorylation of 2 serines at the N-terminus of the inhibitory subunits IB␣, IB␤ or IB⑀. Subsequent IB gets ubiquitinated and degradated by the proteasome. 4 This reaction leaves the active form of NF-B, which is translocated to the nucleus where it binds to regulatory sequences in promoter/enhancer sequences. The mutation or deletion of the 2 serine residues 32 and 36 in IB␣ blocks phosphorylation and degradation of IB␣ protein and results in a superrepressor form of IB␣. 5 Two catalytic subunits, IKK␣ and IKK␤, and a regulatory component, NEMO/IKK␥, have been identified within a large 700 -900 kDa protein complex, which upon stimulation phosphorylates IB␣ at these critical serines. 6 Phosphorylation is followed by an ubiquitination and rapid degradation of IB by a proteasome dependent pathway. 7 This allows the translocation of free active NF-B complexes into the nucleus, where they bind specific DNA motifs in the promoter/enhancer regions of target genes and activate transcription.In this study, we present evidence that the transcription factor NF-B/Rel is constitutively activated in human pancreatic cancer specimen as well as in pancreatic cancer cell lines. NF-B/Rel activity is a result of constitutive IB kinase (IKK) activity and can be inhibited by dominant negative mutants of EGF-R and Ras, the PI3 kinase inhibitor LY294002 or dominant negative mutant Akt kinase. Inhibition of IKK or NF-B/Rel activity induced apoptosis and inhibited anchorage-dependent as well as -independent proliferation of MiaPaCa2 and Panc1 cells. These data demonstrate that NF-B/Rel activity reveals a mitogenic and antiapoptotic role in pancreatic cancer. MATERIAL AND METHODS Tissue samples and immunohistochemistryTissue samples were obtained during surgery on patients with suspected pancreatic cancer and shock frozen in liquid nitrogen. Diagnosis of pancreatic adenocarcinoma was confirmed by an independent pathologist. Sections were fixed with 4% formaldehyde for 15 min and sequentially treated with 0.1% Triton X 100 for 5 min and RNAseA (5 mg/ml) for 30 min at 37°C. Sections were blocked in 2% BSA, 3% goat serum in PBS for 30 min.

. Thus, TGF-␣ is able to promote progression throughout G 1 , but not S phase. Crossbreeding with p53 null mice accelerates tumor development in TGF-␣ transgenic mice dramatically. In tumors developing in these mice, biallelic deletion of Ink4a/Arf or LOH of the Smad4 locus is found suggesting that loci in addition to p53 are involved in antitumor activities. We conclude that these genetic events are critical for pancreatic tumor formation in mice. This model recapitulates pathomorphological features and genetic alterations of the human disease.

Hierarchically organized regulatory proteins form a complex network for expression control of symbiotic and accessory genes in the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum. A genome-wide survey of regulatory interactions was made possible with the design of a custom-made gene chip. Here, we report the first use of the microarray in a comprehensive and complete characterization of the B. japonicum NifA+sigma(54) regulon which forms an important node in the entire network. Comparative transcript profiles of anaerobically grown wild-type, nifA, and rpoN (1/2) mutant cells were complemented with a position-specific frequency matrix-based search for NifA- and sigma(54)-binding sites plus a simple operon definition. One of the newly identified NifA+sigma(54)-dependent genes, fdxN, encodes a ferredoxin required for efficient symbiotic nitrogen fixation, which makes it a candidate for being a direct electron donor to nitrogenase. The fdxN gene has an unconventional, albeit functional sigma(54 )promoter with the dinucleotide GA instead of the consensus GC motif at position -12. A GC-containing mutant promoter and the atypical GA-containing promoter of the wild type were disparately activated. Expression analyses were also carried out with two other NifA+sigma(54) targets (ectC; ahpC). Incidentally, the tiling-like design of the microarray has helped to arrive at completely revised annotations of the ectC- and ahpC-upstream DNA regions, which are now compatible with promoter locations. Taken together, the approaches used here led to a substantial expansion of the NifA+sigma(54 )regulon size, culminating in a total of 65 genes for nitrogen fixation and diverse other processes.

We report a proteomics strategy to both identify and quantify cellular target protein interactions with externally introduced ligands. We determined dissociation constants for target proteins interacting with the ligand of interest by combining quantitative mass spectrometry with a defined set of affinity purification experiments. We demonstrate the general utility of this methodology in interaction studies involving small-molecule kinase inhibitors, a tyrosine-phosphorylated peptide and an antibody as affinity ligands.

NF-κB is regulated by inhibitor proteins (IκBs), which retain NF-κB in the cytoplasm. Signal-induced phosphorylation by the IκB-kinase complex containing the IκB-kinases 1 and 2 (IKK-1/2 or IKK-α/β) and subsequent degradation of the IκB proteins are prerequisites for NF-κB activation. Many signals induce NF-κB, one of them being oncogenic Raf kinase. We investigated whether NF-κB induction is critical for Raf-mediated transformation. Here, we demonstrate that inhibition of NF-κB interferes with transformation by the Raf-oncogene, and we characterized the mechanism of NF-κB induction by activated Raf kinase and the tumor promoter phorbol 12-myristate 13-acetate (PMA). NF-κB activation by PMA and Raf critically depends on the IκB-kinase complex, most notably on IKK-2. A major signaling pathway induced by Raf is the mitogenic cytoplasmic kinase cascade. However, different inhibitors of this cascade do not affect PMA- and Raf-mediated NF-κB activation. Raf does not phosphorylate the IκB-kinase proteins directly. Raf rather synergizes with another membrane shuttle kinase MEKK1, and Raf-mediated activation of NF-κB is blocked by a dominant negative form of MEKK1. These results suggest that Raf induction of NF-κB is relayed by MEKK1, but not by the classical mitogenic cytoplasmic kinase cascade.

No abstract

Activation of the inhibitor of NF-κB kinase/NF-κB (IKK/NF-κB) system and expression of proinflammatory mediators are major events in acute pancreatitis. However, the in vivo consequences of IKK activation on the onset and progression of acute pancreatitis remain unclear. Therefore, we modulated IKK activity conditionally in pancreatic acinar cells. Transgenic mice expressing the reverse tetracycline-responsive transactivator (rtTA) gene under the control of the rat elastase promoter were generated to mediate acinar cell-specific expression of IKK2 alleles. Expression of dominant-negative IKK2 ameliorated cerulein-induced pancreatitis but did not affect activation of trypsin, an initial event in experimental pancreatitis. Notably, expression of constitutively active IKK2 was sufficient to induce acute pancreatitis. This acinar cell-specific phenotype included edema, cellular infiltrates, necrosis, and elevation of serum lipase levels as well as pancreatic fibrosis. IKK2 activation caused increased expression of known NF-κB target genes, including mediators of the inflammatory response such as TNF-α and ICAM-1. Indeed, inhibition of TNF-α activity identified this cytokine as an important effector of IKK2-induced pancreatitis. Our data identify the IKK/NF-κB pathway in acinar cells as being key to the development of experimental pancreatitis and the major factor in the inflammatory response typical of this disease. IntroductionThe NF-κB transcription factors play a prominent role in controlling the integration of innate immunity into the inflammatory response and adaptive immunity. The activation and nuclear translocation of NF-κB induces the expression of a diverse range of proinflammatory genes, including chemokines, cytokines, and cell adhesion molecules, all necessary for an effective defense response to infectious agents. However, failure to terminate or resolve the inflammatory response has detrimental consequences for the organism. As NF-κB is one of the main transcriptional regulators of inflammation, pathological activation of NF-κB is often associated with chronic inflammatory diseases like rheumatoid arthritis, inflammatory bowel disease, asthma, and multiple sclerosis (1-3).NF-κB represents a family of homodimeric and heterodimeric transcription factors composed of 5 members, namely p50, p52, RelA/p65, RelB, and c-Rel. NF-κB is activated by a large number of inducers, including factors critically involved in the inflammatory response such as TNF-α, IL-1β, and microbial products. These factors activate the TNF, IL-1, Nod-like, and Toll-like receptor systems and thereby initiate signaling cascades that converge on the classical NF-κB pathway. This induces the nuclear translocation of NF-κB dimers typically composed of p50 and RelA/p65. The pivotal regulatory step in this pathway is the signal-induced phosphorylation of inhibitor of NF-κB (IκB) proteins, which are mediated by the IκB kinase (IKK) complex. In unstimulated cells, IκB proteins interact with the NF-κB proteins and inhibit their nuclear translo...