• Anti-beta2GPI-antibody-induced endothelial cell gene expression profiling reveals induction of novel pro-inflammatory genes potentially involved in primary antiphospholipid syndrome.

      Hamid, Colleen G.; Norgate, K.; D'Cruz, D.P.; Khamashta, M.A.; Arno, M.; Pearson, J.D.; Frampton, Geoffrey; Murphy, John J. (BMJ Publishing & European League Against Rheumatism, 2007)
      OBJECTIVE: To determine the effects of primary antiphospholipid syndrome (PAPS)-derived anti-beta(2)GPI antibodies on gene expression in human umbilical vein endothelial cells (HUVEC) by gene profiling using microarrays. METHODS: Anti-beta(2)GPI antibodies purified from sera of patients with PAPS or control IgG isolated from normal subjects were incubated with HUVEC for 4 h before isolation of RNA and processing for hybridisation to Affymetrix Human Genome U133A-2.0 arrays. Data were analysed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. For selected genes microarray data were confirmed by real-time PCR analysis or at the protein level by ELISA. RESULTS: A total of 101 genes were found to be upregulated and 14 genes were downregulated twofold or more in response to anti-beta(2)GPI antibodies. A number of novel genes not previously associated with APS were induced, including chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1, the receptors Tenascin C, OLR1, IL-18 receptor 1, and growth factors CSF2, CSF3 IL-6, IL1beta and FGF18. The majority of downregulated genes were transcription factors/signalling molecules including ID2. Quantitative real-time RT-PCR analysis confirmed the microarray results for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C). CONCLUSIONS: This study reveals a complex gene expression response in HUVEC to anti-beta(2)GPI antibodies with multiple chemokines, pro-inflammatory cytokines, pro-thrombotic and pro-adhesive genes regulated by these antibodies in vitro. Some of these newly identified anti-beta(2)GPI antibody-regulated genes could contribute to the vasculopathy associated with this disease.
    • Aspirin and alterations in DNA repair proteins in the SW480 colorectal cancer cell line.

      Dibra, H. K.; Brown, J. E.; Hooley, Paul; Nicholl, I. D. (Spandios Publications, 2010)
      Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of expression of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2, hMSH6 and hPMS2 in DNA MMR proficient cells. We report that treatment of the DNA MMR competent/p53 mutant colorectal cancer cell line SW480 with 1 mM aspirin for 48 h caused changes in mRNA expression of several key genes involved in DNA damage signalling pathways, including a significant down-regulation in transcription of the genes ATR, BRCA1 and MAPK12. Increases in the transcription of XRCC3 and GADD45alpha genes are also reported. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo-protective effect of aspirin in vivo. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45alpha, an increase in XRCC3 encoded protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This is the first report of XRCC3 gene transcription and encoded protein expression being susceptible to exposure to the non-steroidal anti-inflammatory drug, aspirin. Furthermore, this study indicates that alterations in gene transcription seen in microarray studies must be verified at the protein level.
    • Citrullination of histone H3 drives IL-6 production by bone marrow mesenchymal stem cells in MGUS and multiple myeloma

      McNee, G; Eales, KL; Wei, W; Williams, DS; Barkhuizen, A; Bartlett, DB; Essex, S; Anandram, S; Filer, A; Moss, PAH; et al. (Springer Science and Business Media LLC, 2016-07-11)
      © 2017 Macmillan Publishers Limited, part of Springer Nature. Multiple myeloma (MM), an incurable plasma cell malignancy, requires localisation within the bone marrow. This microenvironment facilitates crucial interactions between the cancer cells and stromal cell types that permit the tumour to survive and proliferate. There is increasing evidence that the bone marrow mesenchymal stem cell (BMMSC) is stably altered in patients with MM - a phenotype also postulated to exist in patients with monoclonal gammopathy of undetermined significance (MGUS) a benign condition that precedes MM. In this study, we describe a mechanism by which increased expression of peptidyl arginine deiminase 2 (PADI2) by BMMSCs in patients with MGUS and MM directly alters malignant plasma cell phenotype. We identify PADI2 as one of the most highly upregulated transcripts in BMMSCs from both MGUS and MM patients, and that through its enzymatic deimination of histone H3 arginine 26, PADI2 activity directly induces the upregulation of interleukin-6 expression. This leads to the acquisition of resistance to the chemotherapeutic agent, bortezomib, by malignant plasma cells. We therefore describe a novel mechanism by which BMMSC dysfunction in patients with MGUS and MM directly leads to pro-malignancy signalling through the citrullination of histone H3R26.
    • Elucidation of the mechanisms underlying the angiogenic effects of ginsenoside Rg(1) in vivo and in vitro.

      Yue, Patrick Y. K.; Wong, Daisy Y. L.; Ha, Wai-Yan; Fung, M.C.; Mak, Nai Ki; Yeung, H.W.; Leung, Hei Wun; Chan, Kelvin C.; Liu, Liang; Fan, T. P. David; et al. (Springer Verlag, 2005)
      The major active constituents of ginseng are ginsenosides, and Rg(1) is a predominant compound of the total extract. Recent studies have demonstrated that Rg(1) can promote angiogenesis in vivo and in vitro. In this study, we used a DNA microarray technology to elucidate the mechanisms of action of Rg(1). We report that Rg(1) induces the proliferation of HUVECs, monitored using [(3)H]-thymidine incorporation and Trypan blue exclusion assays. Furthermore, Rg(1) (150-600 nM) also showed an enhanced tube forming inducing effect on the HUVEC. Rg(1) was also demonstrated to promote angiogenesis in an in vivo Matrigel plug assay, and increase endothelial sprouting in the ex vivo rat aorta ring assay. Differential gene expression profile of HUVEC following treatment with Rg(1) revealed the expression of genes related to cell adhesion, migration and cytoskeleton, including RhoA, RhoB, IQGAP1, CALM2, Vav2 and LAMA4. Our results suggest that Rg(1) can promote angiogenesis in multiple models, and this effect is partly due to the modulation of genes that are involved in the cytoskeletal dynamics, cell-cell adhesion and migration.
    • Evaluation of a functional epigenetic approach to identify promoter region methylation in phaeochromocytoma and neuroblastoma

      Margetts, CDE; Morris, M; Astuti, D; Gentle, DC; Cascon, A; McRonald, FE; Catchpoole, D; Robledo, M; Neumann, HPH; Latif, F; et al. (Bioscientifica, 2008-09-01)
      The molecular genetics of inherited phaeochromocytoma have received considerable attention, but the somatic genetic and epigenetic events that characterise tumourigenesis in sporadic phaeochromocytomas are less well defined. Previously, we found considerable overlap between patterns of promoter region tumour suppressor gene (TSG) hypermethylation in two neural crest tumours, neuroblastoma and phaeochromocytoma. In order to identify candidate biomarkers; and epigenetically inactivated TSGs in phaeochromocytoma and neuroblastoma, we characterised changes in gene expression in three neuroblastoma cell lines after treatment with the demethylating agent 5-azacytidine. Promoter region methylation status was then determined for 28 genes that demonstrated increased expression after demethylation. Three genes HSP47, homeobox A9 (HOXA9) and opioid binding protein (OPCML) were methylated in > 10% of phaeochromocytomas (52, 17 and 12% respectively). Two of the genes, epithelial membrane protein 3 (EMP3) and HSP47, demonstrated significantly more frequent methylation in neuroblastoma than phaeochromocytoma. These findings extend epigenotype of phaeochromocytoma and identify candidate genes implicated in sporadic phaeochromocytoma tumourigenesis. © 2008 Society for Endocrinology.
    • Identification of candidate tumour suppressor genes frequently methylated in renal cell carcinoma

      Morris, MR; Ricketts, C; Gentle, D; Abdulrahman, M; Clarke, N; Brown, M; Kishida, T; Yao, M; Latif, F; Maher, ER; et al. (Springer Nature, 2010-02-15)
      Promoter region hyermethylation and transcriptional silencing is a frequent cause of tumour suppressor gene (TSG) inactivation in many types of human cancers. Functional epigenetic studies, in which gene expression is induced by treatment with demethylating agents, may identify novel genes with tumour-specific methylation. We used high-density gene expression microarrays in a functional epigenetic study of 11 renal cell carcinoma (RCC) cell lines. Twenty-eight genes were then selected for analysis of promoter methylation status in cell lines and primary RCC. Eight genes (BNC1, PDLIM4, RPRM, CST6, SFRP1, GREM1, COL14A1 and COL15A1) showed frequent (30% of RCC tested) tumour-specific promoter region methylation. Hypermethylation was associated with transcriptional silencing. Re-expression of BNC1, CST6, RPRM and SFRP1 suppressed the growth of RCC cell lines and RNA interference knock-down of BNC1, SFRP1 and COL14A1 increased the growth of RCC cell lines. Methylation of BNC1 or COL14A1 was associated with a poorer prognosis independent of tumour size, stage or grade. The identification of these epigenetically inactivated candidate RCC TSGs can provide insights into renal tumourigenesis and a basis for developing novel therapies and biomarkers for prognosis and detection. © 2010 Macmillan Publishers Limited.
    • Mechanistic and predictive profiling of 5-Fluorouracil resistance in human cancer cells.

      Wang, Weiguang; Cassidy, James; O'Brien, Vincent; Ryan, Kevin; Collie-Duguid, Elaina (American Association for Cancer Research, 2004)
      Gene expression was analyzed in five pairs of 5-fluorouracil (5-FU) resistant and parental cancer cell lines on DNA microarrays. In unsupervised analysis, a prediction rule was built from the expression profiles of 29 genes, and 5-FU sensitivity class was predicted with 100% accuracy and high predictive strength. In supervised analysis of key 5-FU pathways, expression of 91 genes was associated with 5-FU sensitivity phenotype and segregated samples accordingly in hierarchical analysis. Key genes involved in 5-FU activation were significantly down-regulated (thymidine kinase, 2.9-fold; orotate phosphoribosyltransferase, 2.3-fold; uridine monophosphate kinase, 3.2-fold; pyrimidine nucleoside phosphorylase 3.6-fold) in resistant cells. Overexpression of thymidylate synthase and its adjacent gene, c-Yes, was detected in the resistant cell lines. The mRNA and protein overexpression of nuclear factor kappaB (NFkappaB) p65 and related antiapoptotic c-Flip gene was detected in resistant cells. The 5-FU-resistant cell lines also showed high NFkappaB DNA-binding activity. Cotransfection of NFkappaB p50 and p65 cDNA induced 5-FU resistance in MCF-7 cells. Both NFkappaB- and 5-FU-induced resistant cell lines manifested reduced expression of genes governing G(1)-S and S-phase transition. Expression of genes involved in DNA replication was also down-regulated in resistant cell lines. These findings were highly consistent with the slower growth rate, higher proportion of G(1), and lower proportion of S-phase cells in the resistant cell lines. This phenotype may protect resistant cells from cell death induced by incorporation of 5-FU into DNA chains, by allowing time to repair 5-FU-induced damage. Our findings may provide novel targets for tackling 5-FU resistance.
    • Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.

      Petch, Amelia K.; Sohail, Muhammad; Hughes, Marcus D.; Benter, Ibrahim; Darling, John L.; Southern, Edwin M.; Akhtar, Saghir (Amsterdam: Elsevier, 2003)
      Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3-an antisense ODN complementary to an inaccessible site in EGFR mRNA-were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology.
    • A morphogenetic EphB/EphrinB code controls hepatopancreatic duct formation

      Thestrup, MI; Caviglia, S; Cayuso, J; Heyne, RLS; Ahmad, R; Hofmeister, W; Satriano, L; Wilkinson, DG; Andersen, JB; Ober, EA; et al. (Springer Science and Business Media LLC, 2019-11-19)
      The hepatopancreatic ductal (HPD) system connects the intrahepatic and intrapancreatic ducts to the intestine and ensures the afferent transport of the bile and pancreatic enzymes. Yet the molecular and cellular mechanisms controlling their differentiation and morphogenesis into a functional ductal system are poorly understood. Here, we characterize HPD system morphogenesis by high-resolution microscopy in zebrafish. The HPD system differentiates from a rod of unpolarized cells into mature ducts by de novo lumen formation in a dynamic multi-step process. The remodeling step from multiple nascent lumina into a single lumen requires active cell intercalation and myosin contractility. We identify key functions for EphB/EphrinB signaling in this dynamic remodeling step. Two EphrinB ligands, EphrinB1 and EphrinB2a, and two EphB receptors, EphB3b and EphB4a, control HPD morphogenesis by remodeling individual ductal compartments, and thereby coordinate the morphogenesis of this multi-compartment ductal system.
    • Variation between two near isogenic barley (Hordeum vulgare) cultivars in expression of the B subunit of the vacuolar ATPase in response to salinity.

      Wei, Wenxue; Bilsborrow, Paul E.; Hooley, Paul; Fincham, Daron A.; Forster, Brian P. (Wiley InterScience, 2001)
      A gene encoding the barley vacuolar ATPase subunit B (BSVAP) was differentially expressed between two near isogenic barley cultivars, Golden Promise and Maythorpe. This gene (BSVAP) was isolated by the mRNA differential display technique (DDRT-PCR). BSVAP was salt inducible under long-term salinity stress in the salt sensitive cultivar Maythorpe but less so in the relatively salt tolerant Golden Promise and was more highly expressed under control conditions in Maythorpe. The physiological consequences of altered vacuolar ATPase expression are discussed in relation to the salt sensitivity of Maythorpe.