Resistance to death receptor–mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin's lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain–containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1β–converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-κB. Despite expression of other NF-κB–dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.

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BackgroundMYC is a key transcription factor involved in central cellular processes such as regulation of the cell cycle, histone acetylation and ribosomal biogenesis. It is overexpressed in the majority of human tumors including aggressive B-cell lymphoma. Especially Burkitt lymphoma (BL) is a highlight example for MYC overexpression due to a chromosomal translocation involving the c-MYC gene. However, no genome-wide analysis of MYC-binding sites by chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) has been conducted in BL so far.Methodology/Principal FindingsChIP-Seq was performed on 5 BL cell lines with a MYC-specific antibody giving rise to 7,054 MYC-binding sites after bioinformatics analysis of a total of approx. 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the cell cycle, ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC-binding sites also accumulate in many B-cell relevant genes. To assess the functional consequences of MYC binding, the ChIP-Seq data were supplemented with siRNA- mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in the B-cell function were up-regulated in response to MYC silencing.Conclusion/SignificanceThe 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in BL and shed further light on the enormous complexity of the MYC regulatory network. Especially our observations that (i) many B-cell relevant genes are targeted by MYC and (ii) that MYC down-regulation leads to an up-regulation of B-cell genes highlight an interesting aspect of BL biology.

To investigate the role of amino acid neurotransmitters in the regulation of LH secretion in ovariectomized (ovx) rats with or without estrogen substitution, we measured the release rates of γ-aminobutyric acid (GABA), taurine, glycine, aspartate, glutamate, homocysteic acid, and also of the neurally inactive amino acids serine and glutamine in push-pull perfusate samples of the preoptic/anterior hypothalamic area (PO/AH) collected at 30-min intervals. To achieve this we had to develop a highly sensitive assay utilizing phenylisothiocyanate prederivatization which was followed by HPLC chromatography. In confirmation of our earlier results we observed again a conspicuous drop of preoptic GABA release prior to and during the time of estrogen-induced LH surge. In addition, the release rates of the excitatory amino acid neurotransmitters aspartate and glutamate in the PO/AH increased during this time. Interestingly, also secretion of taurine and glycine was increased during the LH surge, whereas preoptic release rates of serine and glutamine and of homocysteic acid, the putative endogenous ligand of the so-called N-methyl-D-aspartate receptor, remained unchanged. No such changes of amino acid neurotransmitters release rates were observed in ovx rats. This finding underlines that the changes of amino acid secretion in ovx estrogen-primed rats are likely due to the influence of the steroid rather than due to a diurnal rhythm. We conclude that GnRH neurons are under a tonic inhibitory tone exerted by GABA which is relieved during the time of the estrogen-induced LH surge. During this time, aspartate and glutamate may have additional stimulatory effects on GnRH neurons. The unchanged preoptic release rates of the neurally inactive neurotransmitters serine and glutamine give evidence for the selectivity of the observed effects.

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Background:Lung cancer is the leading cause of cancer-related deaths worldwide. The typical and atypical carcinoid (TC and AC), the large-cell neuroendocrine carcinoma (LCNEC) and the small-cell lung cancers (SCLC) are subgroups of pulmonary tumours that show neuroendocrine differentiations. With the rising impact of molecular pathology in routine diagnostics the interest for reliable biomarkers, which can help to differentiate these subgroups and may enable a more personalised treatment of patients, grows.Methods:A collective of 70 formalin-fixed, paraffin-embedded (FFPE) pulmonary neuroendocrine tumours (17 TCs, 17 ACs, 19 LCNECs and 17 SCLCs) was used to identify biomarkers by high-throughput sequencing. Using the Illumina TruSeq Amplicon-Cancer Panel on the MiSeq instrument, the samples were screened for alterations in 221 mutation hot spots of 48 tumour-relevant genes.Results:After filtering >26 000 detected variants by applying strict algorithms, a total of 130 mutations were found in 29 genes and 49 patients. Mutations in JAK3, NRAS, RB1 and VHL1 were exclusively found in SCLCs, whereas the FGFR2 mutation was detected in LCNEC only. KIT, PTEN, HNF1A and SMO were altered in ACs. The SMAD4 mutation corresponded to the TC subtype. We prove that the frequency of mutations increased with the malignancy of tumour type. Interestingly, four out of five ATM-mutated patients showed an additional alteration in TP53, which was by far the most frequently altered gene (28 out of 130; 22%). We found correlations between tumour type and IASLC grade for ATM- (P=0.022; P=0.008) and TP53-mutated patients (P<0.001). Both mutated genes were also associated with lymph node invasion and distant metastasis (P⩽0.005). Furthermore, PIK3CA-mutated patients with high-grade tumours showed a reduced overall survival (P=0.040) and the mutation frequency of APC and ATM in high-grade neuroendocrine lung cancer patients was associated with progression-free survival (PFS) (P=0.020).Conclusions:The implementation of high-throughput sequencing for the analysis of the neuroendocrine lung tumours has revealed that, even if these tumours encompass several subtypes with varying clinical aggressiveness, they share a number of molecular features. An improved understanding of the biology of neuroendocrine tumours will offer the opportunity for novel approaches in clinical management, resulting in a better prognosis and prediction of therapeutic response.

Background and Purpose NF‐κB‐driven inflammation is negatively regulated by the zinc finger protein A20. Gibberellic acid (GA3) is a plant‐derived diterpenoid with documented anti‐inflammatory activity, which is reported to induce A20‐like zinc finger proteins in plants. Here, we sought to investigate the anti‐inflammatory effect of GA3 in airway epithelial cells and determine if the anti‐inflammatory action relates to A20 induction. Experimental Approach Primary nasal epithelial cells and a human bronchial epithelial cell line (16HBE14o‐) were used. Cells were pre‐incubated with GA3, stimulated with Pseudomonas aeruginosa LPS; IL‐6 and IL‐8 release, A20, NF‐κB and IκBα expression were then evaluated. To determine if any observed anti‐inflammatory effect occurred via an A20‐dependent mechanism, A20 was silenced using siRNA. Key Results Cells pre‐incubated with GA3 had significantly increased levels of A20 mRNA (4 h) and protein (24 h), resulting in a significant reduction in IL‐6 and IL‐8 release. This effect was mediated via reduced IκBα degradation and reduced NF‐κB (p65) expression. Furthermore, the anti‐inflammatory action of GA3 was abolished in A20‐silenced cells. Conclusions and Implications We showed that A20 induction by GA3 attenuates inflammation in airway epithelial cells, at least in part through its effect on NF‐κB and IκBα. GA3 or gibberellin‐derived derivatives could potentially be developed into anti‐inflammatory drugs for the treatment of chronic inflammatory diseases associated with A20 dysfunction. Linked Articles This article is part of a themed section on Inflammation: maladies, models, mechanisms and molecules. To view the other articles in this section visit 10.1111/bph.2016.173.issue-4

A20 and TRAF1 are two anti-apoptotic components of the intracellular signalling pathway of the tumour necrosis factor receptor (TNFR) family. Induction of apoptosis seems to be a main function of these receptors. It is astonishing that a member of this family, CD30, is overexpressed by highly proliferating tumours such as Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). It is known that CD30 stimulation leads to the apoptosis of ALCL tumour cells but not of Hodgkin-Reed-Sternberg (HRS) cells. We have already established the overexpression of TRAF1 in HRS cells. In this study we demonstrate that A20 is highly expressed in the HRS cells in 20/22 of cases of classical HL, in 4/4 cases of nodular lymphocyte-predominant HL (NLPHL), and in 2/2 cases of the anaplastic variant of diffuse large B cell lymphoma. In contrast, all other non-Hodgkin lymphomas, including ALCL, revealed either no A20 reactivity, or reactivity in less than 1% of all tumour cells. CD30 stimulation induced A20 and TRAF1 expression. This effect was most prominent in HL and ALCL cell lines with low basal expression levels of these molecules. Immunohistological studies of reactive lymphoid blasts in tonsillar tissue demonstrated that co-expression of CD30, A20, and TRAF1 also occurs in vivo. Cell lines with high basal A20 and TRAF1 expression were resistant to CD30-mediated apoptosis. The sensitivity to CD30-induced apoptosis was increased by inhibition of protein synthesis. TRAF1 transfection decreased CD30-induced apoptosis. Our data suggest that A20 and TRAF1 contribute to apoptosis resistance and, therefore, play an important role in the pathogenesis of classical HL.