Isolation of promoter for cytosolic phospholipase A2 (cPLA2)

Cytosolic phospholipase A2 (cPLA2) releases arachidonic acid from membrane phospholipids and is believed to be the rate-limiting enzyme in the arachidonic acid pathway. We report herein the isolation of a 3 kb fragment of rodent genomic DNA containing part of the first intron, the first exon and 5'-flanking sequence. The start site of transcription was mapped by 5'-rapid amplification of cDNA ends and corroborated by ribonuclease protection assay. The gene has a TATAless promoter with no classical Sp1 binding sites or initiator element. A microsatellite series of CA repeats was noted in the 5'-flanking region of both the rodent and human promoters. Deletion constructs have been analysed for luciferase activity and confirmed promoter activity.

Proteomic identification of plasma proteins as markers of growth promoter abuse in cattle

Growth-promoting agents are continually misused for increasing animal growth and fraudulent gain in the meat industry, yet detection rates from conventional targeted testing for drug residues do not reflect this. This is because testing currently relies on direct detection of drugs or related metabolites and administrators of such compounds can take adaptive measures to avoid detection through the use of endogenous or unknown drugs, and low dose or combined mixtures. New detection methods are needed which focus on the screening of biological responses of an animal to such growth-promoting agents as it has been demonstrated that genomic, proteomic and metabolomics profiles are altered by xenobiotic intake. Therefore, an untargeted proteomics approach using comparative two-dimensional gel electrophoresis (2DE) was carried out to identify putative proteins altered in plasma after treatment with oestradiol, dexamethasone or prednisolone. Twenty-four male cattle were randomly assigned to four groups (n = 6) for experimental treatment over 40 days, namely a control group of non-treated cattle, and three groups administered 17β-oestradiol-3-benzoate (0.01 mg/kg, intramuscular), dexamethasone sodium phosphate (0.7 mg/day, per os) or prednisolone acetate (15 mg/day, per os), respectively. Plasma collected from each animal at day 25 post study initiation was subjected to proteomic analysis by 2DE for comparison of protein expression between treated and untreated animals. Analysis of acquired gel images revealed 22 plasma proteins which differed in expression by more than 50 % (p < 0.05) in treated animals compared to untreated animals. Proteins of interest underwent identification by LC–MS/MS analysis and were found to have associated roles in transport, blood coagulation, immune response and metabolism pathways. In this way, seven proteins are highlighted as novel biomarker candidates including transthyretin which is shown to be significantly increased in all treatment groups compared to control animals and potentially may find use as global markers of suspect anabolic practice.

IGFBP7 promoter methylation and gene expression analysis in prostate cancer

PURPOSE: IGFBP7 belongs to a family of insulin-like growth factor-1 regulatory binding proteins. IGFBP7 hypermethylation is associated with its down-regulation in various carcinomas. In prostate cancer IGFBP7 down-regulation has been widely reported but to our knowledge the mechanisms behind this event are unknown. We performed a denaturing high performance liquid chromatography screening and validation strategy to profile the methylation status of IGFBP7 in prostate cancer.

MATERIALS AND METHODS: We combined denaturing high performance liquid chromatography and bisulfite sequencing to examine IGFBP7 methylation in a panel of prostate cancer cell lines. Quantitative methylation specific polymerase chain reaction was used to determine methylation levels in prostate tissue specimens of primary prostate cancer, histologically benign prostate adjacent to tumor, high grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. IGFBP7 gene expression was measured by quantitative methylation specific polymerase chain reaction in cell lines and tissue specimens.

RESULTS: IGFBP7 was methylated in the 4 prostate cancer cell lines DU145, LNCaP, PC-3 and 22RV1. Quantitative methylation specific polymerase chain reaction analysis revealed that promoter methylation was associated with decreased IGFBP7 expression. Quantitative methylation specific polymerase chain reaction showed that IGFBP7 methylation was more frequently detected in prostate cancer (60% (31/52)) and high grade prostatic intraepithelial neoplasia (40% (6/15)) samples compared to histologically benign prostate adjacent to tumor (10%) and benign prostatic hyperplasia (0%) samples.

CONCLUSIONS: To our knowledge this is the first report of aberrant IGFBP7 promoter hypermethylation and concurrent IGFBP7 gene silencing in prostate cancer cell lines. Results demonstrate that CpG methylation of IGFBP7 may represent a novel biomarker of prostate cancer and pre-invasive neoplasms. Thus, future examination of IGFBP7 methylation and expression in a larger patient cohort, including bodily fluids, is justified to further evaluate its role in a diagnostic and prognostic setting.

In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer

BACKGROUND: Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).

METHODS: Based on an in silico selection process, 13 genes were screened for methylation in CaP cell lines using DHPLC. Quantitative methylation specific PCR was employed to determine methylation levels in prostate tissue specimens (n = 135), representing tumor, histologically benign prostate, high-grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. Gene expression was measured by QRT-PCR in cell lines and tissue specimens.

RESULTS: The promoters of BIK, BNIP3, cFLIP, TMS1, DCR1, DCR2, and CDKN2A appeared fully or partially methylated in a number of malignant cell lines. This is the first report of aberrant methylation of BIK, BNIP3, and cFLIP in CaP. Quantitative methylation analysis in prostate tissues identified 5 genes (BNIP3, CDKN2A, DCR1, DCR2 and TMS1) which were frequently methylated in tumors but were unmethylated in 100% of benign tissues. Furthermore, 69% of tumors were methylated in at least one of the five-gene panel. In the case of all genes, except BNIP3, promoter hypermethylation was associated with concurrent downregulation of gene expression.

CONCLUSION: Future examination of this "CaP apoptotic methylation signature" in a larger cohort of patients is justified to further evaluate its value as a diagnostic and prognostic marker.

Promoter methylation correlates with reduced Smad4 expression in advanced prostate cancer

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor in a wide range of cell types. A transducer of TGF-beta signaling known as Mothers against decapentaplegic homologue 4 (Smad4) is a known tumor suppressor found on chromosome 18q21.1 and is typically inactivated by deletion or mutation in pancreatic and colorectal cancers. The purpose of the article is to investigate Smad4 expression, gene copy number and methylation status in advanced cases of prostate cancer.

METHODS: We have employed Methylation Specific PCR (MSP) to identify methylation sites within the Smad4 promoter and combined this with quantitative real-time PCR to look for correlates between methylation status and Smad4 expression and to examine androgen receptor (AR) expression. Bacterial artificial chromosome-comparative genomic hybridization (BAC-CGH) has been used to look for genomic amplifications and deletions which may also contribute to expression changes.

RESULTS: We fail to find evidence of genomic deletions or amplifications affecting the Smad4 locus on chromosome 18 but show a correlation between promoter methylation and the loss of Smad4 expression in the same material. We confirm that the AR locus on the X chromosome is amplified in 30% of the advanced clinical samples and that this correlates with increased transcript levels as previously reported by other groups.

CONCLUSION: This indicates that epigenetic changes affect the expression of the Smad4 protein in prostate cancer and points to methylation of the promoter as a novel marker of and contributor to the disease warranting further study.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

HNF1B variants associate with promoter methylation and regulate gene networks activated in prostate and ovarian cancer

Two independent regions within HNF1B are consistently identified in prostate and ovarian cancer genome-wide association studies (GWAS); their functional roles are unclear. We link prostate cancer (PC) risk SNPs rs11649743 and rs3760511 with elevated HNF1B gene expression and allele-specific epigenetic silencing, and outline a mechanism by which common risk variants could effect functional changes that increase disease risk: functional assays suggest that HNF1B is a pro-differentiation factor that suppresses epithelial-to-mesenchymal transition (EMT) in unmethylated, healthy tissues. This tumor-suppressor activity is lost when HNF1B is silenced by promoter methylation in the progression to PC. Epigenetic inactivation of HNF1B in ovarian cancer also associates with known risk SNPs, with a similar impact on EMT. This represents one of the first comprehensive studies into the pleiotropic role of a GWAS-associated transcription factor across distinct cancer types, and is the first to describe a conserved role for a multi-cancer genetic risk factor.

Serotonin Transporter Gene Polymorphism and Myocardial Infarction - Etude Cas-te'moins de L'Infarctus du Myocarde (ECTIM)

Background— Depression is a risk factor for myocardial infarction (MI). Selective serotonin reuptake inhibitors reduce this risk. The site of action is the serotonin transporter (SLC6A4), which is expressed in brain and blood cells. A functional polymorphism in the promoter region of the SLC6A4 gene has been described. This polymorphism may be associated with the risk of MI. Methods and Results— The SLC6A4 polymorphism has been investigated by polymerase chain reaction in 671 male patients with MI and in 688 controls from the Etude Cas-Témoins de l’Infarctus du Myocarde (ECTIM) multicentric study. Percentages for LL, LS, and SS genotypes were 35.5%, 45.4%, and 19.1%, respectively, for cases versus 28.1%, 49.1%, and 22.8%, respectively, for controls. S allele frequency was 41.8% and 47.4% for cases and controls, respectively. After adjustment for age and center by using multivariable logistic regression, the odds ratio for MI associated with the LL genotype was 1.40 (95% CI 1.11 to 1.76, P=0.0047). Conclusions— The LL genotype of the SLC6A4 polymorphism is associated with a higher risk of MI. This could be attributable to the effect of the polymorphism on serotonin-mediated platelet activation or smooth muscle cell proliferation or on other risk factors, such as depression or response to stress

Sequence variation in the CHAT locus shows no association with late-onset Alzheimer's disease

There is substantial evidence for a susceptibility gene for late-onset Alzheimer's disease (AD) on chromosome 10. One of the characteristic features of AD is the degeneration and dysfunction of the cholinergic system. The genes encoding choline acetyltransferase (ChAT) and its vesicular transporter (VAChT), CHAT and SLC18A3 respectively, map to the linked region of chromosome 10 and are therefore both positional and obvious functional candidate genes for late-onset AD. We have screened both genes for sequence variants and investigated each for association with late-onset AD in up to 500 late-onset AD cases and 500 control DNAs collected in the UK. We detected a total of 17 sequence variants. Of these, 14 were in CHAT, comprising three non-synonymous variants (D7N in the S exon, A120T in exon 5 and L243F in exon 8), one synonymous change (H547H), nine single-nucleotide polymorphisms in intronic, untranslated or promoter regions, and a variable number of tandem repeats in intron 7. Three non-coding SNPs were detected in SLC18A3. None demonstrated any reproducible association with late-onset AD in our samples. Levels of linkage disequilibrium were generally low across the CHAT locus but two of the coding variants, D7N and A120T, proved to be in complete linkage disequilibrium.

Lack of association between the-2518G/A polymorphism of the MCP-1 gene and ischaemic heart disease: a family-based investigation

Using two recently described family-based tests of association, the possible role of the functional -2518G/A polymorphism in the promoter region of the monocyte chemoattractant protein-1 (MCP-1) gene in the susceptibility to ischaemic heart disease (IHD) was investigated in a well-defined Irish population. One thousand and twelve individuals from 386 families with at least one member prematurely affected with M were, genotyped for the MCP-1 -2518G/A polymorphism. Using the combined transmission disequilibriurn test and the pedigree disequilibriurn test, no association between the MCP-1 -2518G/A polymorphism and M was found. g Our data demonstrate that, in an Irish population, the MCP-1 -2518G/A polymorphism is not strongly associated with M.

Cytokine gene polymorphisms in ischaemic heart disease:Investigation using family based tests of association

Atherosclerosis has an inflammatory basis, with cytokines, cellular adhesion molecules and pro-inflammatory cells having important roles in the initiation and progression of this process. Interleukin (IL) 6, IL-10 and transforming growth factor (TGF) β have been proposed as important modulators of the atherosclerotic process, with IL-6 having a pro-inflammatory, atherogenic effect and IL-10 and TGF-β having anti-inflammatory, protective roles. The possible role of functional polymorphisms in the promoter regions of the IL-6, IL-10 and TGF-β genes in the susceptibility to ischaemic heart disease (IHD) was investigated in a well-defined Irish population using two recently described family-based tests of association. We genotyped 1,012 individuals from 386 families with at least one member prematurely affected with IHD. Using the combined transmission disequilibrium test (TDT)/sib-TDT and the pedigree disequilibrium test, no association between any of the IL-6 -174G/C, IL-10 -1082G/A and TGF-β -509C/T polymorphisms and IHD was found. Our data demonstrate that, in an Irish population, these polymorphisms are not associated with IHD. © Springer-Verlag 2004.

High glucose decreases intracellular glutathione concentrations and upregulates inducible nitric oxide synthase gene expression in intestinal epithelial cells

Diabetes is associated with oxidative stress and increased levels of inflammatory cytokines. The aim of the study was to assess the effects of inflammatory cytokines and oxidative stress associated with raised glucose levels on inducible nitric oxide synthase (iNOS) promoter activity in intestinal epithelial cells. High glucose (25 mmol/l) conditions reduced glutathione (GSH) levels in the human intestinal epithelial cell line, DLD-1. Addition of the antioxidant alpha-lipoic acid resulted in the restoration of GSH levels to normal. Upregulation of basal iNOS promoter activity was observed when cells were incubated in high glucose alone. This effect was significantly reduced by the addition of the antioxidant, alpha-lipoic acid and completely blocked with inhibition of NFkappa B activity. Cytokine stimulation [interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma] induced iNOS promoter activity in all conditions and this was accompanied by an increase in nitric oxide (NO) production. Inhibition of NFkappa-B activity decreased but did not completely inhibit cytokine-induced iNOS promoter activity and subsequent NO production. In conclusion, high glucose-induced iNOS promoter activity is mediated in part through intracellular GSH and NFkappa-B.

Preproendothelin-1 expression in human mesangial cells: evidence for a p38 mitogen-activated protein kinase/protein kinases- C-dependent mechanism

Endothelin-1 (ET-1) has been implicated in the pathogenesis of renal inflammation. This study investigated the mechanisms underlying the synergistic upregulation of preproET-1 gene expression in human mesangial cells after co-stimulation with thrombin and tumor necrosis factor alpha (TNFalpha). Whereas thrombin induced a moderate upregulation of preproET-1 mRNA, co-stimulation with TNFalpha resulted in a strong and protracted upregulation of this mRNA species. Thrombin+TNFalpha-induced upregulation of preproET-1 expression was found to require p38 mitogen-activated protein kinase and protein kinases C, whereas activation of extracellular signal-regulated kinase, c-Jun-N-terminal kinase, or intracellular Ca(2+) release were not required. Actinomycin D chase experiments suggested that enhanced stability of preproET-1 mRNA did not account for the increase in transcript levels. PreproET-1 promoter analysis demonstrated that the 5'-flanking region of preproET-1 encompassed positive regulatory elements engaged by thrombin. Negative modulation of thrombin-induced activation exerted by the distal 5' portion of preproET-1 promoter (-4.4 kbp to 204 bp) was overcome by co-stimulation with TNFalpha, providing a possible mechanism underlying the synergistic upregulation of preproET-1 expression by these two agonists. In conclusion, human mesangial cell expression of preproET-1 may be increased potently in the presence of two common proinflammatory mediators, thereby providing a potential mechanism for ET-1 production in inflammatory renal disease.

BRCA1 Suppresses Osteopontin-mediated Breast Cancer

BRCA1 is a well described breast cancer susceptibility gene thought to be involved primarily in DNA repair. However, mutation within the BRCA1 transcriptional domain is also implicated in neoplastic transformation of mammary epithelium, but responsible mechanisms are unclear. Here we show in a rat mammary model system that wild type (WT) BRCA1 specifically represses the expression of osteopontin (OPN), a multifunctional estrogen-responsive gene implicated in oncogenic transformation, particularly that of the breast. WT.BRCA1 selectively binds OPN-activating transcription factors estrogen receptor alpha, AP-1, and PEA3, inhibits OPN promoter transactivation, and suppresses OPN mRNA and protein both from an endogenous gene and a relevant model inducible gene. WT.BRCA1 also inhibits OPN-mediated neoplastic transformation characterized by morphology change, anchorage-independent growth, adhesion to fibronectin, and invasion through Matrigel. A mutant BRCA1 allele (Mut.BRCA1) associated with familial breast cancer lacks OPN suppressor effects, binds to WT.BRCA1, and impedes WT.BRCA1 suppression of OPN. Stable transfection of rat breast tumor cell lines with Mut.BRCA1 dramatically up-regulates OPN protein and induces anchorage independent growth. In human primary breast cancer, BRCA1 mutation is significantly associated with OPN overexpression. Taken together, these data suggest that BRCA1 mutation may confer increased tissue-specific cancer risk, in part by disruption of BRCA1 suppression of OPN gene transcription.