Characterizing the tumor suppressor activity of FLCN in Birt-Hogg-Dubé syndrome through transcriptiomic and proteomic analysis.

Birt-Hogg-Dubé (BHD) syndrome patients are uniquely susceptible to all renal tumour subtypes. The underlying mechanism of carcinogenesis is unclear. To study cancer development in BHD, we used human proximal kidney (HK2) cells and found that long-term folliculin (FLCN) knockdown was required to increase their tumorigenic potential, forming larger spheroids in non-adherent conditions. Transcriptomic and proteomic analysis uncovered links between FLCN, cell cycle control and the DNA damage response (DDR) machinery. HK2 cells lacking FLCN had an altered transcriptome profile with cell cycle control gene enrichment. G1/S cell cycle checkpoint signaling was compromised with heightened protein levels of cyclin D1 (CCND1) and hyperphosphorylation of retinoblastoma 1 (RB1). A FLCN interactome screen uncovered FLCN binding to DNA-dependent protein kinase (DNA-PK). This novel interaction was reversed in an irradiation-responsive manner. Knockdown of FLCN in HK2 cells caused a marked elevation of γH2AX and RB1 phosphorylation. Both CCND1 and RB1 phosphorylation remained raised during DNA damage, showing an association with defective cell cycle control with FLCN knockdown. Furthermore, Flcn-knockdown C. elegans were defective in cell cycle arrest by DNA damage. This work implicates that long-term FLCN loss and associated cell cycle defects in BHD patients could contribute to their increased risk of cancer.

PIGA Mutations and Glycosylphosphatidylinositol Anchor Dysregulation in Polyposis-Associated Duodenal Tumorigenesis.

The pathogenesis of duodenal tumors in the inherited tumor syndromes familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) is poorly understood. This study aimed to identify genes that are significantly mutated in these tumors and to explore the effects of these mutations. Whole exome and whole transcriptome sequencing identified recurrent somatic coding variants of phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PIGA) in 19/70 (27%) FAP and MAP duodenal adenomas, and further confirmed the established driver roles for APC and KRAS. PIGA catalyzes the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Flow cytometry of PIGA-mutant adenoma-derived and CRISPR-edited duodenal organoids confirmed loss of GPI anchors in duodenal epithelial cells and transcriptional profiling of duodenal adenomas revealed transcriptional signatures associated with loss of PIGA. IMPLICATIONS: PIGA somatic mutation in duodenal tumors from patients with FAP and MAP and loss of membrane GPI-anchors may present new opportunities for understanding and intervention in duodenal tumorigenesis.

Stroma-derived extracellular vesicle mRNA signatures inform histological nature of prostate cancer.

Histological assessment of prostate cancer is the key diagnostic test and can predict disease outcome. This is however an invasive procedure that carries associated risks, hence non-invasive assays to support the diagnostic pathway are much needed. A key feature of disease progression, and subsequent poor prognosis, is the presence of an altered stroma. Here we explored the utility of prostate stromal cell-derived vesicles as indicators of an altered tumour environment. We compared vesicles from six donor-matched pairs of adjacent-normal versus disease-associated primary stromal cultures. We identified 19 differentially expressed transcripts that discriminate disease from normal stromal extracellular vesicles (EVs). EVs isolated from patient serum were investigated for these putative disease-discriminating mRNA. A set of transcripts including Caveolin-1 (CAV1), TMP2, THBS1, and CTGF were found to be successful in discriminating clinically insignificant (Gleason = 6) disease from clinically significant (Gleason > 8) prostate cancer. Furthermore, correlation between transcript expression and progression-free survival suggests that levels of these mRNA may predict disease outcome. Informed by a machine learning approach, combining measures of the five most informative EV-associated mRNAs with PSA was shown to significantly improve assay sensitivity and specificity. An in-silico model was produced, showcasing the superiority of this multi-modal liquid biopsy compared to needle biopsy for predicting disease progression. This proof of concept highlights the utility of serum EV analytics as a companion diagnostic test with prognostic utility, which may obviate the need for biopsy.

Reproductive history determines Erbb2 locus amplification, WNT signalling and tumour phenotype in a murine breast cancer model.

Understanding the mechanisms underlying tumour heterogeneity is key to the development of treatments that can target specific tumour subtypes. We have previously targeted CRE recombinase-dependent conditional deletion of the tumour suppressor genes Brca1, Brca2, p53 (also known as Trp53) and/or Pten to basal or luminal oestrogen receptor-negative (ER-) cells of the mouse mammary epithelium. We demonstrated that both the cell-of-origin and the tumour-initiating genetic lesions cooperate to influence mammary tumour phenotype. Here, we use a CRE-activated HER2 orthologue to specifically target HER2/ERBB2 oncogenic activity to basal or luminal ER- mammary epithelial cells and perform a detailed analysis of the tumours that develop. We find that, in contrast to our previous studies, basal epithelial cells are less sensitive to transformation by the activated NeuKI allele, with mammary epithelial tumour formation largely confined to luminal ER- cells. Histologically, most tumours that developed were classified as either adenocarcinomas of no special type or as metaplastic adenosquamous tumours. The former were typically characterized by amplification of the NeuNT/Erbb2 locus; in contrast, tumours displaying squamous metaplasia were enriched in animals that had been through at least one pregnancy and typically had lower levels of NeuNT/Erbb2 locus amplification but had activated canonical WNT signalling. Squamous changes in these tumours were associated with activation of the epidermal differentiation cluster. Thus, in this model of HER2 breast cancer, cell-of-origin, reproductive history, NeuNT/Erbb2 locus amplification and the activation of specific branches of the WNT signalling pathway all interact to drive inter-tumour heterogeneity.

JMJD6 promotes self-renewal and regenerative capacity of hematopoietic stem cells.

Lifelong multilineage hematopoiesis critically depends on rare hematopoietic stem cells (HSCs) that reside in the hypoxic bone marrow microenvironment. Although the role of the canonical oxygen sensor hypoxia-inducible factor prolyl hydroxylase has been investigated extensively in hematopoiesis, the functional significance of other members of the 2-oxoglutarate (2-OG)-dependent protein hydroxylase family of enzymes remains poorly defined in HSC biology and multilineage hematopoiesis. Here, by using hematopoietic-specific conditional gene deletion, we reveal that the 2-OG-dependent protein hydroxylase JMJD6 is essential for short- and long-term maintenance of the HSC pool and multilineage hematopoiesis. Additionally, upon hematopoietic injury, Jmjd6-deficient HSCs display a striking failure to expand and regenerate the hematopoietic system. Moreover, HSCs lacking Jmjd6 lose multilineage reconstitution potential and self-renewal capacity upon serial transplantation. At the molecular level, we found that JMJD6 functions to repress multiple processes whose downregulation is essential for HSC integrity, including mitochondrial oxidative phosphorylation (OXPHOS), protein synthesis, p53 stabilization, cell cycle checkpoint progression, and mTORC1 signaling. Indeed, Jmjd6-deficient primitive hematopoietic cells display elevated basal and maximal mitochondrial respiration rates and increased reactive oxygen species (ROS), prerequisites for HSC failure. Notably, an antioxidant, N-acetyl-l-cysteine, rescued HSC and lymphoid progenitor cell depletion, indicating a causal impact of OXPHOS-mediated ROS generation upon Jmjd6 deletion. Thus, JMJD6 promotes HSC maintenance and multilineage differentiation potential by suppressing fundamental pathways whose activation is detrimental for HSC function.

Novel pyrrolobenzodiazepine benzofused hybrid molecules inhibit NF-κB activity and synergise with bortezomib and ibrutinib in hematological cancers.

Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are incurable hematological malignancies that are pathologically linked with aberrant NF-κB activation. In this study, we identified a group of novel C8-linked benzofused Pyrrolo[2,1-c][1,4]benzodiazepines (PBD) monomeric hybrids capable of sequence-selective inhibition of NF-κB with low nanomolar LD50 values in CLL (n=46) and MM cell lines (n=5). The lead compound, DC-1-192, significantly inhibited NF-κB DNA binding after just 4h exposure and demonstrating inhibitory effects on both canonical and non-canonical NF-κB subunits. In primary CLL cells, sensitivity to DC-1-192 was inversely correlated with RelA subunit expression (r2=0.2) and samples with BIRC3 or NOTCH1 mutations showed increased sensitivity (P=0.001). RNA-sequencing and gene set enrichment analysis confirmed the over-representation of NF-κB regulated genes in the down-regulated gene list. Furthermore, In vivo efficacy studies in NOD/SCID mice, using a systemic RPMI 8226 human multiple myeloma xenograft model, showed that DC-1-192 significantly prolonged survival (P=0.017). In addition, DC1-192 showed synergy with bortezomib and ibrutinib; synergy with ibrutinib was enhanced when CLL cells were co-cultured on CD40L-expressing fibroblasts in order to mimic the cytoprotective lymph node microenvironment (P = 0.01). Given that NF-κB plays a role in both bortezomib and ibrutinib resistance mechanisms, these data provide a strong rationale for the use of DC-1-192 in the treatment of NF-κB-driven cancers, particularly in the context of relapsed/refractory disease.

Zeb1 modulates hematopoietic stem cell fates required for suppressing acute myeloid leukemia.

Zeb1, a zinc finger E-box binding homeobox epithelial-mesenchymal transition (EMT) transcription factor, confers properties of "stemness," such as self-renewal, in cancer. Yet little is known about the function of Zeb1 in adult stem cells. Here, we used the hematopoietic system as a well-established paradigm of stem cell biology to evaluate Zeb1-mediated regulation of adult stem cells. We employed a conditional genetic approach using the Mx1-Cre system to specifically knock out (KO) Zeb1 in adult hematopoietic stem cells (HSCs) and their downstream progeny. Acute genetic deletion of Zeb1 led to rapid-onset thymic atrophy and apoptosis-driven loss of thymocytes and T cells. A profound cell-autonomous self-renewal defect and multilineage differentiation block were observed in Zeb1-KO HSCs. Loss of Zeb1 in HSCs activated transcriptional programs of deregulated HSC maintenance and multilineage differentiation genes and of cell polarity consisting of cytoskeleton-, lipid metabolism/lipid membrane-, and cell adhesion-related genes. Notably, epithelial cell adhesion molecule (EpCAM) expression was prodigiously upregulated in Zeb1-KO HSCs, which correlated with enhanced cell survival, diminished mitochondrial metabolism, ribosome biogenesis, and differentiation capacity and an activated transcriptomic signature associated with acute myeloid leukemia (AML) signaling. ZEB1 expression was downregulated in AML patients, and Zeb1 KO in the malignant counterparts of HSCs - leukemic stem cells (LSCs) - accelerated MLL-AF9- and Meis1a/Hoxa9-driven AML progression, implicating Zeb1 as a tumor suppressor in AML LSCs. Thus, Zeb1 acts as a transcriptional regulator in hematopoiesis, critically coordinating HSC self-renewal, apoptotic, and multilineage differentiation fates required to suppress leukemic potential in AML.

Sensitivity of human papillomavirus­positive and ­negative oropharyngeal cancer cell lines to ionizing irradiation.

Human papillomavirus­positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) has increased in incidence and has a much better prognosis than HPV­negative (HPV­) OPSCC with radiotherapy alone, but exactly why is unknown. The present study therefore aimed to further examine the sensitivity and possible changes in gene expression of several HPV+ and HPV­ OPSCC, including various novel cell lines, upon ionizing irradiation (IR). Previously established HPV+ UM­SCC­47, UPCI­SCC­90, CU­OP­2, CU­OP­3 and HPV­ UM­SCC­4, UM­SCC­6, UM­SCC­74a, UM­SCC­19 and newly established CU­OP­17 and CU­OP­20, characterised here, were subjected to 0­6 Gy. Surviving fractions of each cell line were tested by clonogenic assays, and irregularities in cell cycle responses were examined by flow cytometry, while changes in gene expression were followed by mRNA sequencing. HPV+ OPSCC cell lines showed greater variation in sensitivity to ionizing irradiation (IR) and tended to be more sensitive than HPV­ OPSCC cell lines. However, their IR sensitivity was not correlated to the proportion of cells in G2 arrest, and HPV­ cell lines generally showed lower increases in G2 after IR. Upon IR with 2 Gy, mRNA sequencing revealed an increase in minor HPV integration sites in HPV+ cell lines, and some changes in gene expression in OPSCC cell lines, but not primarily those associated with DNA repair. To conclude, HPV+ OPSCC cell lines showed greater variation in their sensitivity to IR, with some that were radioresistant, but overall the HPV+ OPSCC group still tended to be more sensitive to IR than the HPV­ OPSCC group. In addition, HPV+ OPSCC lines were more frequently in G2 as compared to HPV­ cell lines, but the increase in G2 arrest upon IR in HPV+ OPSCC was not correlated to sensitivity to IR. Increases in minor HPV integration sites and changes in gene expression were also demonstrated after irradiation with 2 Gy.

APC transcription studies and molecular diagnosis of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is characterised by the development of hundreds to thousands of colorectal adenomas and results from inherited or somatic mosaic variants in the APC gene. Index patients with suspected FAP are usually investigated by APC coding region sequence and dosage analysis in a clinical diagnostic setting. The identification of an APC variant which is predicted to alter protein function enables predictive genetic testing to guide the management of family members. This report describes a 4-generation family with a phenotype consistent with FAP, but in which an APC variant had not been identified, despite testing. To explore this further, quantitative PCR (qPCR) was employed to assess APC transcription, demonstrating reduced levels of APC RNA. Next generation sequencing (NGS) identified the APC 5'UTR/ Exon 1 variant, c.-190 G>A, that had been reported previously in an another FAP family with APC allelic imbalance. Quantitative RNA studies and DNA sequencing of the APC promoters/ Exon 1 may be useful diagnostically for patients with suspected FAP when coding region variants cannot be identified.

Gata2 as a Crucial Regulator of Stem Cells in Adult Hematopoiesis and Acute Myeloid Leukemia.

Subversion of transcription factor (TF) activity in hematopoietic stem/progenitor cells (HSPCs) leads to the development of therapy-resistant leukemic stem cells (LSCs) that drive fulminant acute myeloid leukemia (AML). Using a conditional mouse model where zinc-finger TF Gata2 was deleted specifically in hematopoietic cells, we show that knockout of Gata2 leads to rapid and complete cell-autonomous loss of adult hematopoietic stem cells. By using short hairpin RNAi to target GATA2, we also identify a requirement for GATA2 in human HSPCs. In Meis1a/Hoxa9-driven AML, deletion of Gata2 impedes maintenance and self-renewal of LSCs. Ablation of Gata2 enforces an LSC-specific program of enhanced apoptosis, exemplified by attenuation of anti-apoptotic factor BCL2, and re-instigation of myeloid differentiation--which is characteristically blocked in AML. Thus, GATA2 acts as a critical regulator of normal and leukemic stem cells and mediates transcriptional networks that may be exploited therapeutically to target key facets of LSC behavior in AML.

Correction: Loss of tuberous sclerosis complex 2 sensitizes tumors to nelfinavir-bortezomib therapy to intensify endoplasmic reticulum stress-induced cell death.

This article was originally published under standard licence, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly.

Sensitivity to inhibition of DNA repair by Olaparib in novel oropharyngeal cancer cell lines infected with Human Papillomavirus.

The incidence of Human Papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is increasing rapidly in the UK. Patients with HPV-positive OPSCC generally show superior clinical responses relative to HPV-negative patients. We hypothesised that these superior responses could be associated with defective repair of DNA double strand breaks (DSB). The study aimed to determine whether defective DNA repair could be associated with sensitivity to inhibition of DNA repair using the PARP inhibitor Olaparib. Sensitivity to Olaparib, and induction and repair of DNA damage, were assessed in a panel of 8 OPSCC cell-lines, including 2 novel HPV-positive lines. Effects on cell cycle distribution and levels of PARP1 and p53 were quantified. RNA-sequencing was used to assess differences in activity of DNA repair pathways. Two HPV-positive OPSCC lines were sensitive to Olaparib at potentially therapeutic doses (0.1-0.5 µM). Two HPV-negative lines were sensitive at an intermediate dose. Four other lines, derived from HPV-positive and HPV-negative tumours, were resistant to PARP inhibition. Only one cell-line, UPCISCC90, showed results consistent with the original hypothesis i.e. that in HPV-positive cells, treatment with Olaparib would cause accumulation of DSB, resulting in cell cycle arrest. There was no evidence that HPV-positive tumours exhibit defective repair of DSB. However, the data suggest that a subset of OPSCC may be susceptible to PARP-inhibitor based therapy.

Loss of tuberous sclerosis complex 2 sensitizes tumors to nelfinavir-bortezomib therapy to intensify endoplasmic reticulum stress-induced cell death.

Cancer cells lose homeostatic flexibility because of mutations and dysregulated signaling pathways involved in maintaining homeostasis. Tuberous Sclerosis Complex 1 (TSC1) and TSC2 play a fundamental role in cell homeostasis, where signal transduction through TSC1/TSC2 is often compromised in cancer, leading to aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 hyperactivation increases the basal level of endoplasmic reticulum (ER) stress via an accumulation of unfolded protein, due to heightened de novo protein translation and repression of autophagy. We exploit this intrinsic vulnerability of tumor cells lacking TSC2, by treating with nelvinavir to further enhance ER stress while inhibiting the proteasome with bortezomib to prevent effective protein removal. We show that TSC2-deficient cells are highly dependent on the proteosomal degradation pathway for survival. Combined treatment with nelfinavir and bortezomib at clinically relevant drug concentrations show synergy in selectively killing TSC2-deficient cells with limited toxicity in control cells. This drug combination inhibited tumor formation in xenograft mouse models and patient-derived cell models of TSC and caused tumor spheroid death in 3D culture. Importantly, 3D culture assays differentiated between the cytostatic effects of the mTORC1 inhibitor, rapamycin, and the cytotoxic effects of the nelfinavir/bortezomib combination. Through RNA sequencing, we determined that nelfinavir and bortezomib tip the balance of ER protein homeostasis of the already ER-stressed TSC2-deficient cells in favor of cell death. These findings have clinical relevance in stratified medicine to treat tumors that have compromised signaling through TSC and are inflexible in their capacity to restore ER homeostasis.

Fumarate hydratase is a critical metabolic regulator of hematopoietic stem cell functions.

Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1/Hoxa9-driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation.

IL-10 differentially controls the infiltration of inflammatory macrophages and antigen-presenting cells during inflammation.

The inflammatory activation and recruitment of defined myeloid populations is essential for controlling the bridge between innate and adaptive immunity and shaping the immune response to microbial challenge. However, these cells exhibit significant functional heterogeneity and the inflammatory signals that differentially influence their effector characteristics are poorly characterized. In this study, we defined the phenotype of discrete subsets of effective antigen-presenting cells (APCs) in the peritoneal cavity during peritonitis. When the functional properties of these cells were compared to inflammatory monocyte-derived macrophages we noted differential responses to the immune-modulatory cytokine IL-10. In contrast to the suppressive actions of IL-10 on inflammatory macrophages, the recruitment of APCs was relatively refractory and we found no evidence for selective inhibition of APC differentiation. This differential response of myeloid cell subsets to IL-10 may thus have limited impact on development of potentially tissue-damaging adaptive immune responses, while restricting the magnitude of the inflammatory response. These findings may have clinical relevance in the context of peritoneal dialysis patients, where recurrent infections are associated with immune-mediated membrane dysfunction, treatment failure, and increased morbidity.

mRNA sequencing of novel cell lines from human papillomavirus type-16 related vulval intraepithelial neoplasia: consequences of expression of HPV16 E4 and E5.

Vulval intraepithelial neoplasia is a precursor of vulval cancer and is commonly caused by infection with Human Papillomavirus (HPV). Development of topical treatments for vulval intraepithelial neoplasia requires appropriate in vitro models. This study evaluated the feasibility of primary culture of vulval intraepithelial neoplasia biopsy tissue to produce cell lines for use as in vitro models. A potentially immortal cell line was produced which gave rise to three monoclonal lines. These lines were characterized for HPV genomic integration and for viral gene expression using ligation-mediated PCR and quantitative PCR. Distinct patterns of viral integration and gene expression were observed among the three lines. Integration and expression data were validated using deep sequencing of mRNA. Gene ontology analyses of these data also demonstrated that expression of the HPV16 E4 and E5 proteins resulted in substantial changes in the composition of the cell membrane and extracellular space, associated with alterations in cell adhesion and differentiation. These data illustrate the diverse patterns of HPV gene expression potentially present within a single lesion. The derived cell lines provide useful models to investigate the biology of vulval intraepithelial neoplasia and the interactions between different HPV gene products and potential therapeutic agents.

Proteomics analysis of bladder cancer exosomes.

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function.

Adenosine regulates thrombomodulin and endothelial protein C receptor expression in folliculostellate cells of the pituitary gland.

Adenosine stimulates the release of interleukin 6 (IL-6) and vascular endothelial growth factor from folliculostellate cells of the anterior pituitary gland indicating that such cells are also involved in the communication between the immune and endocrine systems during stress and inflammation. In order to understand the precise actions of adenosine on folliculostellate cells, DNA microarray analysis was used to determine global changes in gene expression. Hierarchical clusters revealed, of the genes that had altered expression, the majority were suppressed and many, such as B cell translocation gene 2 and cyclin-dependent kinase inhibitor 2b were related to cell cycle arrest or inhibition of proliferation. Several of the up-regulated genes were associated with cytokine signalling or membrane receptor activity. The most notable of these being IL-6, sulfiredoxin 1, endothelial protein C receptor (EPCR) and thrombomodulin (THBD) which can all play a role in controlling inflammation. The EPCR and THBD pathway is well known in anti-coagulation but also has anti-inflammatory and anti-apoptotic properties. Up-regulation of EPCR and THBD in folliculostellate cells was confirmed by qRT-PCR and western blotting analysis and their expression were also demonstrated in many of the hormone-secreting cells of the anterior pituitary gland. Our findings suggest that adenosine can stimulate expression of stress and inflammation related genes from folliculostellate cells of the anterior pituitary gland. These genes include EPCR and THBD, neither of which has been previously identified in the pituitary gland.

Microarray analysis of senescent vascular smooth muscle cells: A link to atherosclerosis and vascular calcification.

Little is known about the senescent phenotype of human vascular smooth muscle cells (VSMCs) and the potential involvement of senescent VSMCs in age-related vascular disease, such as atherosclerosis. As such, VSMCs were grown and characterised in vitro to generate senescent VSMCs needed for microarray analysis (Affymetrix). Comparative analysis of the transcriptome profiles of early (14 CPD) and late (39-42 CPD) passage VSMCs found a total of 327 probesets called as differentially expressed: 149 are up-regulated in senescence and 178 repressed (p-value<0.5%, minimum effect size of at least 2-fold differential regulation, explore data at http://www.madras.cf.ac.uk/vsmc). Data mining shows a differential regulation of genes at senescence associated with the development of atherosclerosis and vascular calcification. These included genes with roles in inflammation (IL1beta, IL8, ICAM1, TNFAP3, ESM1 and CCL2), tissue remodelling (VEGF, VEGFbeta, ADM and MMP14) and vascular calcification (MGP, BMP2, SPP1, OPG and DCN). The microarray data for IL1beta, IL8 and MGP were validated by either, ELISA, Western blot analysis or RT-PCR. These data thus provide the first evidence for a role of VSMC senescence in the development of vascular calcification and provides further support for the involvement of senescent VSMCs in the progression of atherosclerosis.

Thyrotropin receptor activation increases hyaluronan production in preadipocyte fibroblasts: contributory role in hyaluronan accumulation in thyroid dysfunction.

The thyrotropin receptor (TSHR) is expressed during lineage-specific differentiation (e.g. adipogenesis) and is activated by TSH, thyroid-stimulating antibodies, and gain-of-function mutations (TSHR*). Comparison of gene expression profiles of nonmodified human preadipocytes (n = 4) with the parallel TSHR* population revealed significant up-regulation of 27 genes including hyaluronan (HA) synthases (HAS) 1 and 2. The array data were confirmed by quantitative PCR of HAS1 and HAS2 and enzyme-linked immunosorbent assay measurement of HA; all values were significantly increased (p < 0.03) in TSHR*-expressing preadipocytes (n = 10). Preadipocytes (n = 8) treated with dibutyryl (db)-cAMP display significantly increased HAS1 and HAS2 transcripts, HAS2 protein, and HA production (p < 0.02). HAS1 or HAS2 small interfering RNA treatment of db-cAMP-stimulated preadipocytes (n = 4) produced 80% knockdown in HAS1 or 61% knockdown in HAS2 transcripts (compared with scrambled), respectively; the corresponding HA production was reduced by 49 or 38%. Reporter assays using A293 cells transfected with HAS1 promoter-driven plasmids containing or not containing the proximal CRE and treated with db-cAMP revealed that it is functional. Chromatin immunoprecipitation, using a cAMP-responsive element-binding protein antibody, of db-cAMP-treated preadipocytes (n = 4) yielded products for HAS1 and HAS2 with relative fold increases of 3.3 +/- 0.8 and 2.6 +/- 0.9, respectively. HA accumulates in adipose/connective tissues of patients with thyroid dysfunction. We investigated the contributions of TSH and thyroid-stimulating antibodies and obtained small (9-24%) but significant (p < 0.02) increases in preadipocyte HA production with both ligands. Similar results were obtained with a TSHR monoclonal antibody lacking biological activity (p < 0.05). We conclude that TSHR activation is implicated in HA production in preadipocytes, which, along with thyroid hormone level variation, explains the HA overproduction in thyroid dysfunction.