Effects on prostate cancer cells of targeting RNA polymerase III.

RNA polymerase (pol) III occurs in two forms, containing either the POLR3G subunit or the related paralogue POLR3GL. Whereas POLR3GL is ubiquitous, POLR3G is enriched in undifferentiated cells. Depletion of POLR3G selectively triggers proliferative arrest and differentiation of prostate cancer cells, responses not elicited when POLR3GL is depleted. A small molecule pol III inhibitor can cause POLR3G depletion, induce similar differentiation and suppress proliferation and viability of cancer cells. This response involves control of the fate-determining factor NANOG by small RNAs derived from Alu short interspersed nuclear elements. Tumour initiating activity in vivo can be reduced by transient exposure to the pol III inhibitor. Untransformed prostate cells appear less sensitive than cancer cells to pol III depletion or inhibition, raising the possibility of a therapeutic window.

Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP-activated whole-cell chloride currents in human bronchial epithelial cells.

Chloride channels are known to play critical physiological roles in many cell types. Here, we describe the expression of anion channels using RNA Seq in primary cultures of human bronchial epithelial cells (hBECs). Chloride intracellular channel (CLIC) family members were the most abundant chloride channel transcripts, and CLIC1 showed the highest level of expression. In addition, we characterize the chloride currents in hBECs and determine how inhibition of CLIC1 via pharmacological and molecular approaches impacts these. We demonstrate that CLIC1 is able to modulate cyclic AMP-induced chloride currents and suggest that CLIC1 modulation could be important for chloride homeostasis in this cell type.

The Ser82 RAGE Variant Affects Lung Function and Serum RAGE in Smokers and sRAGE Production In Vitro.

INTRODUCTION: Genome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor (AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model. METHODS: Immunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified. RESULTS: Immunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV1, FEV1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production. CONCLUSIONS: This study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying COPD particularly for carriers of this AGER polymorphism.

Identifying and testing candidate genetic polymorphisms in the irritable bowel syndrome (IBS): association with TNFSF15 and TNFα.

OBJECTIVES: The postinfectious irritable bowel syndrome (PI-IBS) suggests that impaired resolution of inflammation could cause IBS symptoms. The authors hypothesised that polymorphisms in genes whose expression were altered by gastroenteritis might be linked to IBS with diarrhoea (IBS-D) which closely resembles PI-IBS. DESIGN: Part 1: 25 healthy volunteers (HVs), 21 patients 6 months after Campylobacter jejuni infection, 37 IBS-D and 19 IBS with constipation (IBS-C) underwent rectal biopsy for gene expression analysis and peripheral blood mononuclear cell cytokine production assessment. Part 2: Polymorphisms in genes whose expression was altered in Part 1 were assessed in 179 HV, 179 IBS-D, 122 IBS-C and 41 PI-IBS. RESULTS: Part 1: Mucosal expression of seven genes was altered in IBS: CCL11, CCL13, Calpain 8 and TNFSF15 increased while NR1D1, GPR161 and GABRE decreased with similar patterns after infection with C jejuni. Part 2: The authors assessed 21 known single nucleotide polymorphisms (SNPs) in these seven genes and one SNP in each of the TNFα and IL-10 genes. Three out of five TNFSF15 SNPs (rs6478108, rs6478109 and rs7848647) showed reduced minor allele frequency (MAF) (0.28, 0.27 and 0.27) in subjects with IBS-D compared with HV (0.38, 0.36 and 0.37; p=0.007, 0.015 and 0.007, respectively) confirming others recent findings. The authors also replicated the previously reported association of the TNFα SNP rs1800629 with PI-IBS which showed an increase in the MAF at 0.30 versus 0.19 for HV (p=0.04). CONCLUSION: IBS-D and PI-IBS patients are associated with TNFSF15 and TNFα genetic polymorphisms which also predispose to Crohn's disease suggesting possible common underlying pathogenesis.

Impaired uptake of serotonin by platelets from patients with irritable bowel syndrome correlates with duodenal immune activation.

BACKGROUND & AIMS: Patients with irritable bowel syndrome with diarrhea (IBS-D) have increased mucosal serotonin (5-hydroxytryptamine [5-HT]) availability, possibly because immune activation reduces activity of the 5-HT transporter (SERT). We investigated the relationship between mucosal and platelet SERT and immune activation of the duodenal mucosa in patients with IBS-D. METHODS: We quantified mucosal intraepithelial lymphocytes (IELs), mast cells, and enterochromaffin cells in blood samples, measured levels of SERT messenger RNA (mRNA) in mucosal samples, and assessed platelet uptake of 5-HT and platelet membrane binding of (3)H-paroxetine in samples from 29 healthy volunteers (HVs), 20 patients with IBS-D, and 20 untreated patients with celiac disease. RESULTS: Patients with IBS-D or celiac disease had increased numbers of IELs and mast cells compared with HVs (both P < .001). Levels of SERT mRNA were reduced in the mucosa of patients with IBS-D or celiac disease and were inversely correlated with numbers of IELs (r = -0.72, P < .0001). Uptake of 5-HT by platelets from patients with IBS-D or celiac disease was reduced (mean, 17.1 ± 3.5 and 28.3 ± 4.1 nmol·min(-1)·mg(-1), respectively) compared with HVs (50.8 ± 8.0 nmol·min(-1)·mg(-1), P < .01 and P = .05, respectively). Binding of paroxetine to membranes of platelets from patients with IBS-D (median [interquartile range], 226 [92-405] fmol/mg protein) was significantly greater than that from HVs (109 [69-175] fmol/mg protein) and correlated inversely with platelet uptake of 5-HT (r = -0.62, P = .03). Tryptase release from incubated biopsy samples was significantly increased in patients with IBS-D (2.2 [0.42-3.5] vs 0.50 [0.25-0.86] ng·mL(-1)·mg(-1) for HVs; P = .03). CONCLUSIONS: Platelet SERT is reduced in IBS-D and associated with reduced levels of SERT mRNA and duodenal immune activation.

Functional polymorphism and differential regulation of CYSLTR1 transcription in human airway smooth muscle and monocytes.

Cysteinyl leukotrienes play an important role in the pathophysiology of many inflammatory disorders, including asthma. The aim of this study was to characterize the mechanisms underlying transcriptional regulation of the human cysteinyl leukotriene receptor 1 (hCYSLTR1) gene. 5'RACE was performed on human airway smooth muscle (HASM) and peripheral blood mononuclear cells. A1128-bp region of the hCYSLTR1 main putative promoter was screened for polymorphisms by sequencing of 48 individuals. Luciferase reporter gene assays were performed using fragments of the core promoter (232 bp to 1128 bp) in HASM and THP1 cells. Three hCYSLTR1 transcripts were found, one representing 90% of all messenger RNA identified. The genomic location of the transcription start sites suggested there are two putative hCYSLTR1 promoters. The majority of the transcriptional activity of the main putative promoter was detected between -232 and -679 bp. Four singlenucleotide polymorphisms in strong linkage disequilibrium were found in the region studied: -561 (rs7066737), -642 (rs2806489), -781 (rs2637204), and -940 (rs321029), with three haplotypes observed. In THP1 cells, the G allele (-642) caused a twofold decrease in luciferase expression compared to the Aallele. These data suggest that the majority of hCYSLTR1 transcripts in HASM and monocytes arise from a single promoter located immediately upstream of the 5\' untranslated region, although rarer transcripts can also occur. This study also raises the possibility that cell-type-dependent differences in transcriptional activity caused by the presence of specific haplotypes within the main CYSLTR1 promoter may be a predictor of disease risk or treatment response.