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Real-time Reverse Transcriptase-polymerase Chain Reaction (real-time + reverse_transcriptase-polymerase_chain_reaction)
Kinds of Real-time Reverse Transcriptase-polymerase Chain Reaction Selected AbstractsHP24 MICRORNA EXPRESSION PROFILES IN BARRETT'S OESOPHAGUSANZ JOURNAL OF SURGERY, Issue 2007D. I. Watson Purpose The genetic changes that drive the metaplastic change from squamous oesophagus (NO) towards Barrett's oesophagus (BO) and cancer are unclear. microRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression and contribute to cellular differentiation and identity. We sought to determine the role of miRNAs in BO. Methodology Biopsies of NO, BO and cardia were taken from 7 patients and RNA was extracted. miRNA expression profiles of 300 miRNAs were determined by microarray. Guided by the array results, real-time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) for 8 selected miRNAs enabled their expression to be studied in tissues from another 15 patients. Results Array data revealed that 39 miRNAs were significantly differentially expressed between NO, BO and cardia. A tissue-specific expression profile was confirmed by RT-PCR, with miR-21, 143, 145, 194 and 215 significantly up regulated in BO and cardia (columnar) vs. NO (squamous). A trend towards increased miR-21 expression from NO to BO and adenocarcinoma was observed (p = 0.1). Interestingly, high expression of miR-143, 194 and 215 was seen in BO vs. NO (p < 0.0001), but with subsequent downregulation in cancers (p = 0.1). In contrast, miR-203 and 205 were highly expressed in NO and low in BO and cardia. A database search revealed that these miRNAs potentially target (proto-)oncogenes and tumour suppressor genes. Conclusions Differences in miRNA expression are present between NO, BO, cardia and cancer. Deregulation of certain miRNAs, and their predicted effect on the expression of target genes, might contribute to the metaplastic and neoplastic process in the oesophagus and could serve as novel biomarkers to classify diseased tissues. [source] Constitutive expression of the FK506 binding protein 51 (FKBP51) in bone marrow cells and megakaryocytes derived from idiopathic myelofibrosis and non-neoplastic haematopoiesisEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2004Oliver Bock Abstract: Objectives:, Overexpression of FK506 binding protein 51 (FKBP51) in megakaryocytic progenitor cells generated from purified CD34+ cells in patients with idiopathic myelofibrosis (IMF) has been demonstrated. It has been suggested that FKBP51 is involved in the dysregulation of the apoptotic programme with consecutive prolongation of cell survival. The knowledge of FKBP51 and its expression in bone marrow cells and mature megakaryocytes in non-neoplastic haematopoiesis and IMF is sparse. Methods:, To evaluate a potential overexpression of FKBP51 in patients with IMF (n = 37) compared with non-neoplastic haematopoiesis (n = 31), total bone marrow cells as well as single megakaryocytes, isolated by laser microdissection, were quantitatively analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). By applying immunohistochemistry, FKBP51 gene expression was correlated with staining pattern and cellular localisation of the corresponding FKBP51 protein. Results:, We demonstrated that FKBP51 is constitutively expressed in non-neoplastic haematopoiesis. FKBP51 gene expression by total bone marrow cells as well as megakaryocytes was not significantly different in IMF. FKBP51 protein expression could be localised to myeloid progenitor cells as well as megakaryocytes. In particular, megakaryocytes were stained almost exclusively nuclear for FKBP51. No differences in expression patterns between both IMF and control cases could be demonstrated. Conclusions:, For the first time, FKBP51 expression, in particular gene expression and subcellular localization was described in bone marrow cells of non-neoplastic and neoplastic haematopoiesis grown in vivo. We conclude that FKBP51 could be temporarily overexpressed in megakaryocytic progenitors rather than contribute to the accumulation of mature megakaryocytes in IMF. [source] Expression of multidrug resistance-associated protein 1 in invasive ovarian carcinoma: implication for prognosisHISTOPATHOLOGY, Issue 6 2009Areeg Faggad Aims:, Multidrug resistance is a major impediment in chemotherapeutic treatment of ovarian carcinoma patients. The aim of this study was to investigate the expression of multidrug resistance-associated protein 1 (MRP1) and to assess the possible associations with clinicopathological variables and patient outcome in primary ovarian carcinoma. Methods and results:, Tumour specimens from 129 patients were obtained before chemotherapy and analysed by immunohistochemistry on tissue microarrays, and by real-time reverse transcriptase-polymerase chain reaction on RNA extracted from formalin-fixed paraffin-embedded tissue specimens using a new technique. Significantly increased MRP1 protein expression was observed in high-grade tumours (P = 0.005) and advanced International Federation of Gynaecology and Obstetrics stages (P = 0.036). On univariate Kaplan,Meier analysis, patients with higher expression of MRP1 protein had significantly decreased overall survival (P = 0.006). On multivariate Cox regression analysis, MRP1 protein expression retained its significance as an independent negative prognostic marker for overall survival (hazard ratio = 6.52, P = 0.003). Furthermore, MRP1 expression correlated with topoisomerase II, expression both at mRNA and protein level (P < 0.001 and P = 0.023, respectively). Conclusion:, In summary, in patients with primary ovarian cancer, overexpression of MRP1 is an adverse marker for patient outcome and cancer aggressiveness. Our data provide a translational basis for further clinical studies on the predictive value of MRP1 expression for response to chemotherapy. [source] Down-regulation of ATBF1 is a major inactivating mechanism in hepatocellular carcinomaHISTOPATHOLOGY, Issue 5 2008C J Kim Aims:, ,-Fetoprotein (AFP) is frequently detected in hepatocellular carcinomas (HCCs) and AT motif binding factor 1 (ATBF1) down-regulates AFP gene expression in hepatic cells. The ATBF1 gene also inhibits cell growth and differentiation, and altered gene expression is associated with malignant transformation. The aim was to investigate the potential role of the ATBF1 gene in HCCs. Methods and results:, Somatic mutations, allelic loss and hypermethylation of the ATBF1 gene were analysed in 76 sporadic HCCs. The level of ATBF-1 mRNA expression was analysed using quantitative real-time reverse transcriptase-polymerase chain reaction. Genetic studies of the ATBF1 gene revealed absence of somatic mutation in the hotspot region and 15 (25%) of 60 informative cases showed allelic loss at the ATBF1 locus. Hypermethylation in the intron 1 region of the ATBF1 gene was detected in only one case. Interestingly, ATBF1 mRNA expression in HCCs was significantly reduced in 55 (72.4%) samples compared with the corresponding surrounding liver tissues. Reduced expression was not statistically associated with clinicopathological parameters including stage, histological grade, infective virus type, and serum ,-fetoprotein level. Conclusions:, The ATBF1 gene may contribute to the development of HCCs via transcriptional down-regulation of mRNA expression, but not by genetic or epigenetic alterations. [source] Gambogic acid induces apoptosis by regulating the expression of Bax and Bcl-2 and enhancing caspase-3 activity in human malignant melanoma A375 cellsINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 2 2009Xiaoyuan Xu Objectives, To investigate the effect of a Chinese traditional medicine, gambogic acid (GA), on human malignant melanoma (MM) A375 cells and to study the mechanism of apoptosis induced by GA. Methods, A375 cells were treated with GA at different doses and for different times, and their proliferation and viability were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induced by GA in A375 cells was observed by annexin-V/propidium iodide doubling staining flow cytometry assay and Hoechst staining. To further determine the molecular mechanism of apoptosis induced by GA, the changes in expression of Bcl-2 and Bax were detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, and caspase-3 activity was measured by fluorescence resonance energy transfer (FRET) probe. Results, After incubation with GA, A375 cell proliferation was dramatically inhibited in a dose-dependent manner. After these cells had been exposed to GA for 24, 36 and 48 h, the IC50 values were 1.57 ± 0.05, 1.31 ± 0.20, and 1.12 ± 0.19 µg/mL, respectively. Treatment of A375 cells with GA (2.5,7.5 µg/mL) for 36 h resulted in an increased number of early apoptotic cells, which ranged from 27.6% to 41.9%, in a dose-dependent manner, compared with only 3.5% apoptotic cells in the non-GA-treated group. An increase in Bax and decrease in Bcl-2 expression were found by real-time RT-PCR and Western blot. Caspase-3 activity was increased in a dose-dependent manner, observed by FRET probe. Conclusion, GA can inhibit the proliferation of A375 cells and induce their apoptosis, which may be related to the up-regulation of the Bax/Bcl-2 ratio and caspase-3 activity. [source] Periodontal therapy alters gene expression of peripheral blood monocytesJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 9 2007Panos N. Papapanou Abstract Aims: We investigated the effects of periodontal therapy on gene expression of peripheral blood monocytes. Methods: Fifteen patients with periodontitis gave blood samples at four time points: 1 week before periodontal treatment (#1), at treatment initiation (baseline, #2), 6-week (#3) and 10-week post-baseline (#4). At baseline and 10 weeks, periodontal status was recorded and subgingival plaque samples were obtained. Periodontal therapy (periodontal surgery and extractions without adjunctive antibiotics) was completed within 6 weeks. At each time point, serum concentrations of 19 biomarkers were determined. Peripheral blood monocytes were purified, RNA was extracted, reverse-transcribed, labelled and hybridized with AffymetrixU133Plus2.0 chips. Expression profiles were analysed using linear random-effects models. Further analysis of gene ontology terms summarized the expression patterns into biologically relevant categories. Differential expression of selected genes was confirmed by real-time reverse transcriptase-polymerase chain reaction in a subset of patients. Results: Treatment resulted in a substantial improvement in clinical periodontal status and reduction in the levels of several periodontal pathogens. Expression profiling over time revealed more than 11,000 probe sets differentially expressed at a false discovery rate of <0.05. Approximately 1/3 of the patients showed substantial changes in expression in genes relevant to innate immunity, apoptosis and cell signalling. Conclusions: The data suggest that periodontal therapy may alter monocytic gene expression in a manner consistent with a systemic anti-inflammatory effect. [source] Gene Expression Profiles of Intracellular and Membrane Progesterone Receptor Isoforms in the Mediobasal Hypothalamus During Pro-OestrusJOURNAL OF NEUROENDOCRINOLOGY, Issue 12 2009B. Liu Progesterone action is mediated by its binding to specific receptors. Two progesterone receptor (PR) isoforms (PRA and PRB), three membrane progesterone receptor (mPR) subtypes (mPR,, mPR, and mPR,) and at least one progesterone membrane-binding protein [PR membrane component 1 (PRmc1)] have been identified in reproductive tissues and brain of various species. In the present study, we examined gene expression patterns for PR isoforms, mPR subtypes and PRmc1 in the rat mediobasal hypothalamus (MBH) during pro-oestrus. The mRNA level for each receptor subtype was quantified by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) at the time points: 13.00 h on dioestrous day 2; 09.00, 13.00, 17.00 and 22.00 h on pro-oestrus; and 13.00 h on oestrus. For PR, one primer set amplified PRA+PRB, whereas a second primer set amplified PRB. As expected, PRA+PRB mRNA expression was greater than PRB in MBH tissue. PRB mRNA levels increased throughout the day on pro-oestrus, with the highest levels being observed at 17.00 h. PRB mRNA levels in the MBH were increased by 2.4- and 3.0-fold at 13.00 and 17.00 h, respectively, on pro-oestrus compared to 13.00 h on dioestrous day 2. There were differential mRNA expression levels for mPRs and PRmc1 in the MBH, with the highest expression for PRmc1 and the lowest for mPR,. The mPR, mRNA contents at 13.00 and 17.00 h on pro-oestrus were increased by 1.5-fold compared to that at 13.00 h on dioestrous day 2. The mPR, mRNA levels at 13.00 and 17.00 h on pro-oestrus were 2.5- and 2.4-fold higher compared to that at 13.00 h on dioestrous day 2, respectively. PRA+PRB, mPR, and PRmc1 mRNA levels did not vary on pro-oestrus. These findings suggest that the higher expression of PRB, mPR, and mPR, in the MBH on pro-oestrous afternoon may influence both genomic and nongenomic mechanisms of progesterone action during the critical pre-ovulatory period. [source] Pituitary mRNA Expression of the Growth Hormone Axis in the 1-Year-Old Intrauterine Growth Restricted RatJOURNAL OF NEUROENDOCRINOLOGY, Issue 8 2006T. Prins Intrauterine growth restriction (IUGR) is one of the major causes of short stature in childhood. Abnormalities in the growth hormone (GH) axis have frequently been observed in children who are born intrauterine growth restricted and GH treatment is effective to improve final height. However, the way that the GH axis is involved is not fully understood. Previously, when investigating the effect of IUGR on the central somatotrophic axis, a hypothalamic effect was discovered with elevated somatostatin and decreased neuropeptide Y mRNA expression levels, whereas serum GH and insulin-like growth factor I (IGFI) were unaltered. These findings were thought to indicate a hypothalamic alteration of the GH axis due to IUGR, probably to compensate pituitary output, thereby normalising peripheral values of GH and IGFI. Therefore, the present study aimed to evaluate the effect of IUGR on the pituitary GH axis in this rat model. Pups from rats that underwent bilateral uterine artery ligation at day 17 of pregnancy were studied. Pituitary glands were collected from 1-year-old offspring for quantitative measurements of GH, GH-receptor (GH-R), GH-releasing hormone receptor (GHRH-R), somatostatin receptor subtype 2 and 5, IGFI and IGFI receptor mRNA levels using a real-time reverse transcriptase-polymerase chain reaction. In addition, liver GH-R and IGFI mRNA expression levels were measured and a radioimmunoassay was performed to determine serum IGFI levels. In the IUGR rat, levels of pituitary GH, GH-R and GHRH-R relative gene expression (RGE) were increased. No differences were found in the RGE level of all other pituitary growth factors, liver GH-R and IGFI, and serum IGFI concentration between IUGR and control rats. The present data show that intrauterine growth failure leads to changes in the pituitary that might counterbalance the effects found previously in the hypothalamus. [source] Demonstration of Postsynaptic Receptor Plasticity in an Amphibian Neuroendocrine InterfaceJOURNAL OF NEUROENDOCRINOLOGY, Issue 11 2002B. G. Jenks Abstract Pituitary pars intermedia melanotrope cells are often used as a model to study mechanisms of neuroendocrine integration. In the amphibian Xenopus laevis, the synthesis and release of ,-melanophore-stimulating hormone (,-MSH) from these cells is a dynamic process dependent upon the colour of background. In animals on a black background, there is a higher level of synthesis and secretion of ,-MSH than in animals on a white background, and, consequently, there is skin darkening in animals on a black background. The melanotropes are innervated by hypothalamic neurones that produce neuropeptide Y (NPY), a peptide that inhibits ,-MSH secretion via the NPY Y1 receptor. The inhibitory neurones have a higher expression of NPY in animals adapted to a white background and both the size and the number of inhibitory synapses on the melanotrope cells are enhanced. The purpose of the present study was to determine if this presynaptic plasticity displayed by the inhibitory neurones is reciprocated by postsynaptic plasticity (i.e. if there is an enhanced expression of the Y1 receptor in melanotropes of animals adapted to a white background). For this purpose quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the level of Y1 receptor mRNA in melanotropes of animals undergoing the process of background adaptation. The results showed that there is a higher Y1 receptor mRNA expression in melanotropes of white-adapted animals. We conclude that the inhibitory neuroendocrine interface in the Xenopus pars intermedia displays postsynaptic plasticity in response to changes of background colour. To our knowledge, this is the first demonstration of a physiological environmental change leading to changes in postsynaptic receptor expression in a fully identified vertebrate neuroendocrine reflex. [source] Analysis of neuronal gene expression with laser capture microdissectionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2002Valerie A.M. Vincent Abstract The brain is a heterogeneous tissue in which the numbers of neurons, glia, and other cell types vary among anatomic regions. Gene expression studies performed on brain homogenates yield results reflecting mRNA abundance in a mixture of cell types. Therefore, a method for quantifying gene expression in individual cell populations would be useful. Laser capture microdissection (LCM) is a new technique for obtaining pure populations of cells from heterogeneous tissues. Most studies thus far have used LCM to detect DNA sequences. We developed a method to quantify gene expression in hippocampal neurons from mouse brain using LCM and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). This method was optimized to permit histochemical or immunocytochemical visualization of nerve cells during LCM while minimizing RNA degradation. As an example, gene expression was quantified in hippocampal neurons from the Tg2576 mouse model for Alzheimer's disease. © 2002 Wiley-Liss, Inc. [source] Enhanced ability of regulatory T cells in chronic hepatitis C patients with persistently normal alanine aminotransferase levels than those with active hepatitisJOURNAL OF VIRAL HEPATITIS, Issue 12 2009I. Itose Summary., In hepatitis C virus (HCV) infection, the Th1-type immune response is involved in liver injury. A predominance of immunosuppressive regulatory T cells (Treg) is hypothesized in patients with persistently normal alanine aminotransferase (PNALT). Our aim was to clarify the role of Treg in the pathogenesis of PNALT. Fifteen chronically HCV-infected patients with PNALT, 21 with elevated ALT (CH) and 19 healthy subjects (HS) were enrolled. We determined naturally-occurring Treg (N-Treg) as CD4+CD25high+FOXP3+ T cells. The expression of FOXP3 and CTLA4 in CD4+CD25high+ cells was quantified by real-time reverse transcriptase-polymerase chain reaction. Bulk or CD25-depleted CD4+ T cells cultured with HCV-NS5 loaded dendritic cells were assayed for their proliferation and cytokine release. We examined CD127,CD25,FOXP3+ cells as distinct subsets other than CD25+ N-Treg. The frequencies of N-Treg in patients were significantly higher than those in HS. The FOXP3 and CTLA4 transcripts were higher in PNALT than those in CH. The depletion of CD25+ cells enhanced HCV-specific T cell responses, showing that co-existing CD25+ cells are suppressive. Such inhibitory capacity was more potent in PNALT. The frequency of CD4+CD127,CD25,FOXP3+ cells was higher in CH than those in PNALT. Treg are more abundant in HCV-infected patients, and their suppressor ability is more potent in patients with PNALT than in those with active hepatitis. [source] Rapamycin and CCI-779 inhibit the mammalian target of rapamycin signalling in hepatocellular carcinomaLIVER INTERNATIONAL, Issue 1 2010Ivan Chun-Fai Hui Abstract Background: The mammalian target of rapamycin (mTOR), which phosphorylates p70S6K and 4EBP1 and activates the protein translation process, is upregulated in cancers and its activation may be involved in cancer development. Aims: In this study, we investigated the tumour-suppressive effects of rapamycin and its new analogue CCI-779 on hepatocellular carcinoma (HCC). Methods: Rapamycin and its new analogue CCI-779 were applied to treat HCC cells. Cell proliferation, cell cycle profile and tumorigenicity were analysed. Results: In human HCCs, we observed frequent (67%, 37/55) overexpression of mTOR transcripts using real-time reverse transcriptase-polymerase chain reaction. Upon drug treatment, PLC/PRF/5 showed the greatest reduction in cell proliferation using the colony formation assay, as compared with HepG2, Hep3B and HLE. Rapamycin was a more potent antiproliferative agent than CCI-779 in HCC cell lines. Proliferation assays by cell counting showed that the IC50 value of rapamycin was lower than that of CCI-779 in PLC/PRF/5 cells. Furthermore, flow cytometric analysis showed that both drugs could arrest HCC cells in the G1 phase but did not induce apoptosis of these cells, suggesting that these mTOR inhibitors are cytostatic rather than cytotoxic. Upon rapamycin and CCI-779 treatment, the phosphorylation level of mTOR and p70S6K in HCC cell lines was significantly reduced, indicating that both drugs can suppress mTOR activity in HCC cells. In addition, both drugs significantly inhibited the growth of xenografts of PLC/PRF/5 cells in nude mice. Conclusions: Our findings indicate that rapamycin and its clinical analogue CCI-779 possess tumour-suppressive functions towards HCC cells. [source] Up-regulation of proproliferative genes and the ligand/receptor pair placental growth factor and vascular endothelial growth factor receptor 1 in hepatitis C cirrhosisLIVER INTERNATIONAL, Issue 7 2007Xiao X. Huang Abstract Background/Aims: Cirrhosis can lead to hepatocellular carcinoma (HCC). Non-diseased liver and hepatitis C virus (HCV)-associated cirrhosis with or without HCC were compared. Method: Proliferation pathway genes, immune response genes and oncogenes were analysed by a quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining. Results: Real-time RT-PCR showed up-regulation of genes in HCV cirrhosis including the proliferation-associated genes bone morphogenetic protein 3 (BMP3), placental growth factor 3 (PGF3), vascular endothelial growth factor receptor 1 (VEGFR1) and soluble VEGFR1, the oncogene FYN, and the immune response-associated genes toll-like receptor 9 (TLR9) and natural killer cell transcript 4 (NK4). Expressions of TLR2 and the oncogenes B-cell CLL/lymphoma 9 (BCL9) and PIM2 were decreased in HCV cirrhosis. In addition, PIM2 and TLR2 were increased in HCV cirrhosis with HCC compared with HCV cirrhosis. The ligand/receptor pair PGF and VEGFR1 was intensely expressed by the portal tract vascular endothelium. VEGFR1 was expressed in reactive biliary epithelial structures in fibrotic septum and in some stellate cells and macrophages. Conclusion: PGF and VEGFR1 may have an important role in the pathogenesis of the neovascular response in cirrhosis. [source] Expression of Cyclooxygenase-1 and -2 in the Porcine Endometrium during the Oestrous Cycle and Early PregnancyREPRODUCTION IN DOMESTIC ANIMALS, Issue 3 2006A Blitek Contents Cyclooxygenase (COX) is the rate-limiting enzyme that catalyses the initial step in prostaglandins (PGs) production. In the present studies, endometrial COX-1 and COX-2 expression throughout the oestrous cycle and early pregnancy was analysed in pigs using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. There were no changes in messenger RNA (mRNA) and protein expression for COX-1 in cyclic pigs. In pregnant animals, mRNA levels of this enzyme increased on days 22,25 (p < 0.001). However, no upregulation of COX-1 protein was detected. Quantification of COX-2 mRNA expression during the oestrous cycle revealed significant increases on days 10,12 and 14 (p < 0.001 and p < 0.01 vs days 2,4, respectively). Protein levels were also increased on day 14 when compared with days 2,12 and 18,20 after oestrus. In pregnant animals, the patterns of both COX-2 mRNA and protein expression were similar. Messenger RNA levels were higher on days 16 and 22,25 (p < 0.01 vs day 10). Moreover, the protein content tended to increase on days 16 and 22,25. COX-1 and COX-2 were localized in the luminal and glandular epithelium as well as in the uterine stroma. In contrast to COX-1, a positive immunostaining reaction for COX-2 was detected only on days 12,16 after ovulation and on days 14,16 of pregnancy. In conclusion, these results indicate specific patterns of COX-1 and COX-2 expression in the porcine endometrium throughout the oestrous cycle and early pregnancy. COX-2 rather than COX-1 seems to be the primary enzyme responsible for modulated PGs production at the time of luteolysis in cyclic and during implantation in pregnant animals. [source] Local matrix metalloproteinase 2 gene knockdown in balloon-injured rabbit carotid arteries using nonviral-small interfering RNA transfectionTHE JOURNAL OF GENE MEDICINE, Issue 1 2009Hanna Hlawaty Abstract Background Small interfering RNA (siRNA) delivery is a promising approach for the treatment of cardiovascular diseases. Matrix metalloproteinase (MMP) 2 over-expression in the arterial wall has been implicated in restenosis after percutaneous coronary intervention, as well as in spontaneous atherosclerotic plaque rupture. We hypothesized that in vivo local delivery of siRNA targeted at MMP2 (MMP2-siRNA) in the balloon-injured carotid artery of hypercholesterolemic rabbits may lead to inhibition of MMP2 expression. Methods Two weeks after balloon injury, 5 µmol/l of Tamra-tagged MMP2-siRNA, scramble siRNA or saline was locally injected in the carotid artery and incubated for 1 h. Results Fluorescent microscopy studies showed the circumferential uptake of siRNA in the superficial layers of neointimal cells. MMP2 mRNA levels, measured by the real-time reverse transcriptase-polymerase chain reaction, was decreased by 79 ± 25% in MMP2-siRNA- versus scramble siRNA-transfected arteries (p < 0.05). MMP2 activity, measured by gelatin zymography performed on the conditioned media of MMP2-siRNA versus scramble siRNA transfected arteries, decreased by 53 ± 29%, 50 ± 24% and 46 ± 14% at 24, 48 and 72 h, respectively (p < 0.005 for all). No effect was observed on MMP9, pro-MMP9 and TIMP-2 levels. Conclusions The results obtained in the present study suggest that significant inhibition of gene expression can be achieved with local delivery of siRNA in the arterial wall in vivo. Copyright © 2008 John Wiley & Sons, Ltd. [source] Rhinovirus infection-induced alteration of tight junction and adherens junction components in human nasal epithelial cellsTHE LARYNGOSCOPE, Issue 2 2010Nam-Kyung Yeo MD Abstract Objectives/Hypothesis: Manifestations of rhinovirus (RV) infections include mucus overproduction, increased vascular permeability, and secondary bacterial infection. These effects may reflect disrupted epithelial barrier functions, which are mainly regulated by intercellular junctions, referred to as tight junctions (TJs) and adherens junctions (AJs). The objective of this study was to investigate changes in the components of TJs (ZO-1, occluding, and claudin-1) and AJs (E-cadherin) after RV infection in cultured nasal epithelial cells. Methods: Primary human nasal epithelial cells grown at an air-liquid interface were infected apically with RV. RV-induced changes in the expression of epithelial TJ and AJ proteins were determined using real-time reverse transcriptase-polymerase chain reaction, confocal microscopy, and Western blot analyses. Functional changes in the integrity of junctional proteins were assessed by measuring transepithelial resistance (TER) using a voltmeter. Results: RV infection decreased mRNA levels of ZO-1, occludin, claudin-1, and E-cadherin to 64.2%, 51.8%, 56.2%, and 56.3%, respectively, of those in controls (P < .05). Decreases in ZO-1, occludin, claudin-1, and E-cadherin protein levels in RV-infected cells were evident in immunofluorescent confocal microscopic images. Expression levels of these proteins were also lower in the RV-infected group in Western blot analyses. RV infection reduced the mean TER from 143.1 ,/cm2 (controls) to 122.6 ,/cm2. Conclusions: RV infection decreased the expression of TJ and AJ components and reduced TER in primary cultured human nasal epithelial cells, indicating that RV infection may exert a harmful effect on nasal epithelial barrier function. Laryngoscope, 2010 [source] Molecular characterization of upper urinary tract tumoursBJU INTERNATIONAL, Issue 6 2010Laura Izquierdo OBJECTIVES To assess gene-expression patterns of BIRC5, FGFR3, IGF2, KRT20, UPK2, EBF1, CDH1, FXYD3, HTERT, TP53, AGR2, HER2 and VEGF, widely known markers of bladder urothelial carcinoma (UC) in upper tract UC, and to determine their value as prognostic factors of tumour progression and cancer-specific survival. PATIENTS AND METHODS The study included 83 formalin-fixed paraffin-embedded tissue specimens (68 and 15 from patients with UTUC and controls, respectively) collected between 1990 and 2004. Thirteen bladder cancer-related genes were selected from previous reports and analysed by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) in all samples. RESULTS Six genes were over-expressed (BIRC5, FGFR3, KRT20, UPK2, FXYD3 and hTERT) and three under-expressed (AGR2, TP53 and VEGF) in the tumour group (P < 0.05). For four genes (IGF2, EBF1, CDH1 and HER2) there was no statistically significant difference between the tumour and control groups. Overall, 21 patients developed tumour progression and 13 died from UTUC after a mean follow-up of 35.24 months. The 5-year disease-free progression and cancer-specific survival rates were 65.8% and 72.9%, respectively. In a multivariate regression analysis, the independent predictive variable for tumour progression and cancer-specific survival was pathological stage (hazard ratio 3.60, P < 0.001; and 3.73, P < 0.005, respectively), but none of the studied genes were identified as prognostic factors of tumour progression or cancer-specific survival. CONCLUSIONS Our data suggest that bladder cancer and UTUC share some characteristics, but have differences in gene expression. None of BIRC5, FGFR3, IGF2, KRT20, UPK2, EBF1, CDH1, FXYD3, HTERT, TP53, AGR2, HER2 and VEGF were correlated either tumour progression or survival. [source] Wilms tumor gene protein 1 is associated with ovarian cancer metastasis and modulates cell invasionCANCER, Issue 7 2008Maria V. Barbolina PhD Abstract BACKGROUND Although metastatic disease is the primary cause of death from epithelial ovarian carcinoma, to the authors' knowledge the cellular mechanisms that regulate intraperitoneal metastasis are largely unknown. Metastasizing ovarian carcinoma cells encounter a collagen-rich microenvironment because the submesothelial matrix is comprised mainly of interstitial collagens Types I and III. METHODS Immunohistochemistry using primary and metastatic ovarian carcinoma samples was employed to detect expression of Wilms tumor gene protein 1 (WT1). Three-dimensional (3D) collagen culture, real-time reverse transcriptase-polymerase chain reaction, and immunofluorescent staining were used to evaluate changes in WT1 RNA and protein expression in response to 3D collagen culture. Boyden chamber invasion assay, scratch-wound motility assay, and Western blot analysis were used to establish the function of WT1 in ovarian carcinoma cells. RESULTS To model intraperitoneal invasion in vitro, ovarian cancer cells were cultured in a 3D collagen microenvironment. 3D collagen culture resulted in robust induction of WT1 at the mRNA and protein levels. WT1 expression was prevalent in primary ovarian tumors and was retained in paired peritoneal metastases. Functional studies supported a role for WT1 in intraperitoneal invasion, because siRNA knockdown of WT1 expression reduced the ability of ovarian cancer cells to invade 3D collagen gels. CONCLUSIONS The data from the current study identify a novel regulatory mechanism for the control of WT1 expression and provide evidence for a functional role of WT1 protein in the control of cellular invasive activity. Cancer 2008. ©2008 American Cancer Society. [source] Differential Effects of Partial Hepatectomy and Carbon Tetrachloride Administration on Induction of Liver Cell Foci in a Model for Detection of Initiation ActivityCANCER SCIENCE, Issue 10 2001Hiroki Sakai Differential effects of partial hepatectomy (PH) and carbon tetrachloride (CC14) administration on induction of glutathione S-transferase placental form (GST-P)-positive foci were investigated in a model for detection of initiation activity. Firstly, we surveyed cell proliferation kinetics and fluctuation in cytochrome P450 (CYP) mRNA levels by means of relative-quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and CYP 2E1 apoprotein amount by immuno-blotting (experiment I) after PH or CC14 administration. Next, to assess the interrelationships among cell proliferation, fluctuation of CYPs after PH or CC14 administration and induction of liver cell foci, the non-hepatocarcinogen, 1,2-dimethylhydrazine (DMH) was administered to 7-week-old male F344 rats and initiated populations were selected using the resistant hepatocyte model (experiment II). In experiment I, the values of all CYP isozyme mRNAs after PH or CC14 administration were drastically decreased at the 12-h tune point. From 72 h, mRNAs for all CYP isozymes began increasing, with complete recovery after 7 days. The CYP 2E1 apoprotein content in the PH group fluctuated weakly, whereas in the CC14 group it had decreased rapidly after 12 h and was still low at the 48 h point. In experiment II, induction of GST-P-positive foci was related to cell kinetics in the PH group, with about a 6-h time lag between tune for carcinogen administration giving greatest induction of GST-P-positive foci and peaks in bromodeoxyuridine (BrdU) labeling, presumably due to the necessity for bioactivation of DMH. With CC14 administration, induction of foci appeared dependent on the recovery of CYP 2E1. In conclusion, PH was able to induce cell proliferation with maintenance of CYP 2E1, therefore being advantageous for induction of liver cell foci in models to detect initiation activity. [source] | |||||||||||||