Patients with human severe combined immunodeficiency (SCID) can be divided into those with B lymphocytes (B+ SCID) and those without (B- SCID). Although several genetic causes are known for B+ SCID, the etiology of B- SCID has not been defined. Six of 14 B- SCID patients tested were found to carry a mutation of the recombinase activating gene 1 (RAG-1), RAG-2, or both. This mutation resulted in a functional inability to form antigen receptors through genetic recombination and links a defect in one of the site-specific recombination systems to a human disease.
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.
The expression of a variety of cytoprotective genes is regulated by short cis-acting elements in their promoters, called antioxidant response elements (AREs). A central regulator of ARE-mediated gene expression is the NF-E2-related factor 2 (Nrf2). Nrf2/ARE-regulated genes are crucial for the maintenance of cellular integrity. Hepatitis C virus inhibits the induction of ARE-regulated genes, but neither induction nor inhibition of ARE-regulated gene expression affects HCV replication directly. In HCV-replicating cells the core protein triggers the delocalization of sMaf proteins from the nucleus to the replicon complex. Here sMafs bind to NS3. The extranuclear sMaf proteins prevent Nrf2 from entry in the nucleus and thereby inhibit the induction of Nrf2/ARE-regulated genes. This results in the decreased expression of cytoprotective genes. Consistent with this finding, the elimination of ROI is impaired in HCV-replicating cells as demonstrated by elevated protein oxidation or 8-OH-dG formation, reflecting DNA damage. In conclusion, these data identified a novel mechanism of Nrf2 regulation and suggest that the HCV-dependent inhibition of Nrf2/ARE-regulated genes confers to the HCV-associated pathogenesis by elevation of intracellular ROI that affect integrity of the host genome and regenerative processes.
Hepatitis C virus (HCV)2 infection results in chronic hepatitis in more than 70% of infected individuals. At present more than 170 million people are persistently infected with HCV worldwide.
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