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Sample records for lines expressing increased

  1. Increased IMP dehydrogenase gene expression in solid tumor tissues and tumor cell lines

    SciTech Connect

    Collart, F.R.; Chubb, C.B.; Mirkin, B.L.; Huberman, E.

    1992-07-10

    IMP dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this possibility, we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results are consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.

  2. Increased Expression of Several Collagen Genes is Associated with Drug Resistance in Ovarian Cancer Cell Lines.

    PubMed

    Januchowski, Radosław; Świerczewska, Monika; Sterzyńska, Karolina; Wojtowicz, Karolina; Nowicki, Michał; Zabel, Maciej

    2016-01-01

    Ovarian cancer is the most lethal gynaecological cancer. The main reason for the high mortality among ovarian cancer patients is the development of drug resistance. The expression of collagen genes by cancer cells can increase drug resistance by inhibiting the penetration of the drug into the cancer tissue as well as increase apoptosis resistance. In this study, we present data that shows differential expression levels of collagen genes and proteins in cisplatin- (CIS), paclitaxel- (PAC), doxorubicin- (DOX), topotecan- (TOP), vincristine- (VIN) and methotrexate- (MTX) resistant ovarian cancer cell lines. Quantitative real-time polymerase chain reactions were performed to determine the mRNA levels. Protein expression was detected using Western blot and immunocytochemistry assays. In the drug resistant cell lines, we observed the upregulation of eight collagen genes at the mRNA level and based on these expression levels, we divided the collagen genes into the following three groups: 1. Genes with less than a 50-fold increase in expression: COL1A1, COL5A2, COL12A1 and COL17A1. 2. Genes with greater than a 50-fold increase in expression: COL1A2, COL15A1 and COL21A1. 3. Gene with a very high level of expression: COL3A1. Expression of collagen (COL) proteins from groups 2 and 3 were also confirmed using immunocytochemistry. Western blot analysis showed very high expression levels of COL3A1 protein, and immunocytochemistry analysis showed the presence of extracellular COL3A1 in the W1TR cell line. The cells mainly responsible for the extracellular COL3A1 production are aldehyde dehydrogenase-1A1 (ALDH1A1) positive cells. All correlations between the types of cytostatic drugs and the expression levels of different COL genes were studied, and our results suggest that the expression of fibrillar collagens may be involved in the TOP and PAC resistance of the ovarian cancer cells. The expression pattern of COL genes provide a preliminary view into the role of these proteins in

  3. Increased Expression of Several Collagen Genes is Associated with Drug Resistance in Ovarian Cancer Cell Lines

    PubMed Central

    Januchowski, Radosław; Świerczewska, Monika; Sterzyńska, Karolina; Wojtowicz, Karolina; Nowicki, Michał; Zabel, Maciej

    2016-01-01

    Ovarian cancer is the most lethal gynaecological cancer. The main reason for the high mortality among ovarian cancer patients is the development of drug resistance. The expression of collagen genes by cancer cells can increase drug resistance by inhibiting the penetration of the drug into the cancer tissue as well as increase apoptosis resistance. In this study, we present data that shows differential expression levels of collagen genes and proteins in cisplatin- (CIS), paclitaxel- (PAC), doxorubicin- (DOX), topotecan- (TOP), vincristine- (VIN) and methotrexate- (MTX) resistant ovarian cancer cell lines. Quantitative real-time polymerase chain reactions were performed to determine the mRNA levels. Protein expression was detected using Western blot and immunocytochemistry assays. In the drug resistant cell lines, we observed the upregulation of eight collagen genes at the mRNA level and based on these expression levels, we divided the collagen genes into the following three groups: 1. Genes with less than a 50-fold increase in expression: COL1A1, COL5A2, COL12A1 and COL17A1. 2. Genes with greater than a 50-fold increase in expression: COL1A2, COL15A1 and COL21A1. 3. Gene with a very high level of expression: COL3A1. Expression of collagen (COL) proteins from groups 2 and 3 were also confirmed using immunocytochemistry. Western blot analysis showed very high expression levels of COL3A1 protein, and immunocytochemistry analysis showed the presence of extracellular COL3A1 in the W1TR cell line. The cells mainly responsible for the extracellular COL3A1 production are aldehyde dehydrogenase-1A1 (ALDH1A1) positive cells. All correlations between the types of cytostatic drugs and the expression levels of different COL genes were studied, and our results suggest that the expression of fibrillar collagens may be involved in the TOP and PAC resistance of the ovarian cancer cells. The expression pattern of COL genes provide a preliminary view into the role of these proteins in

  4. Neurotoxic Methamphetamine Doses Increase LINE-1 Expression in the Neurogenic Zones of the Adult Rat Brain

    PubMed Central

    Moszczynska, Anna; Flack, Amanda; Qiu, Ping; Muotri, Alysson R.; Killinger, Bryan A.

    2015-01-01

    Methamphetamine (METH) is a widely abused psychostimulant with the potential to cause neurotoxicity in the striatum and hippocampus. Several epigenetic changes have been described after administration of METH; however, there are no data regarding the effects of METH on the activity of transposable elements in the adult brain. The present study demonstrates that systemic administration of neurotoxic METH doses increases the activity of Long INterspersed Element (LINE-1) in two neurogenic niches in the adult rat brain in a promoter hypomethylation-independent manner. Our study also demonstrates that neurotoxic METH triggers persistent decreases in LINE-1 expression and increases the LINE-1 levels within genomic DNA in the striatum and dentate gyrus of the hippocampus, and that METH triggers LINE-1 retrotransposition in vitro. We also present indirect evidence for the involvement of glutamate (GLU) in LINE-1 activation. The results suggest that LINE-1 activation might occur in neurogenic areas in human METH users and might contribute to METH abuse-induced hippocampus-dependent memory deficits and impaired performance on several cognitive tasks mediated by the striatum. PMID:26463126

  5. Neurotoxic Methamphetamine Doses Increase LINE-1 Expression in the Neurogenic Zones of the Adult Rat Brain.

    PubMed

    Moszczynska, Anna; Flack, Amanda; Qiu, Ping; Muotri, Alysson R; Killinger, Bryan A

    2015-10-14

    Methamphetamine (METH) is a widely abused psychostimulant with the potential to cause neurotoxicity in the striatum and hippocampus. Several epigenetic changes have been described after administration of METH; however, there are no data regarding the effects of METH on the activity of transposable elements in the adult brain. The present study demonstrates that systemic administration of neurotoxic METH doses increases the activity of Long INterspersed Element (LINE-1) in two neurogenic niches in the adult rat brain in a promoter hypomethylation-independent manner. Our study also demonstrates that neurotoxic METH triggers persistent decreases in LINE-1 expression and increases the LINE-1 levels within genomic DNA in the striatum and dentate gyrus of the hippocampus, and that METH triggers LINE-1 retrotransposition in vitro. We also present indirect evidence for the involvement of glutamate (GLU) in LINE-1 activation. The results suggest that LINE-1 activation might occur in neurogenic areas in human METH users and might contribute to METH abuse-induced hippocampus-dependent memory deficits and impaired performance on several cognitive tasks mediated by the striatum.

  6. Azidothymidine and cisplatin increase p14ARF expression in OVCAR-3 ovarian cancer cell line

    SciTech Connect

    Vaskivuo, Liisa; Rysae, Jaana; Koivuperae, Johanna; Myllynen, Paeivi; Vaskivuo, Tommi; Chvalova, Katerina; Serpi, Raisa; Savolainen, Eeva-Riitta; Puistola, Ulla; Vaehaekangas, Kirsi . E-mail: kirsi.vahakangas@uku.fi

    2006-10-01

    p14{sup ARF} tumor suppressor protein regulates p53 by interfering with mdm2-p53 interaction. p14{sup ARF} is activated in response to oncogenic stimuli but little is known of the responses of endogenous p14{sup ARF} to different types of cellular stress or DNA damage. Azidothymidine (AZT) is being tested in several clinical trials as an enhancer of anticancer chemotherapy. However, the knowledge of the relationship between AZT and cellular pathways, e.g. p53 pathway, is very limited. In this study, we show that AZT, cisplatin (CDDP) and docetaxel (DTX) all induce unique molecular responses in OVCAR-3 ovarian carcinoma cells carrying a mutated p53, while in A2780, ovarian carcinoma and MCF-7 breast carcinoma cells with wild type p53, all of these drugs cause similar p53 responses. We found that endogenous p14{sup ARF} protein in OVCAR-3 cells is down-regulated by DTX but induced by AZT and a short CDDP pulse treatment. In HT-29 colon carcinoma cells with a mutated p53, all treatments down-regulated p14{sup ARF} protein. Both CDDP and AZT increased the expression of p14ARF mRNA in OVCAR-3 cells. Differences in cell death induced by these drugs did not explain the differences in protein and mRNA expressions. No increase in the level of either c-Myc or H-ras oncoproteins was seen in OVCAR-3 cells after AZT or CDDP-treatment. These results suggest that p14{sup ARF} can respond to DNA damage without oncogene activation in cell lines without functional p53.

  7. Protection against UVA-induced photooxidative damage in mammalian cell lines expressing increased levels of metallothionein

    SciTech Connect

    Dudek, E.J. Illinois Inst. of Tech., Chicago, IL . Dept. of Biology); Peak, J.G.; Peak, M.J. ); Roth, R.M. . Dept. of Biology)

    1990-01-01

    Metallothionein (MT) is an endogenous low molecular weight protein that is inducible in a variety of eukaryotic cells and has the ability to selectivity bind heavy metal ions such as zinc and the cadmium. Although the exact physiological role of MT is still not understood, there is strong evidence that MT is involved in providing cellular resistance against the damaging effects of heavy metals and in the regulation of intracellular zinc and copper. Recently, it has been demonstrated that MT can scavenge radiation-induced reactive oxygen intermediates in vitro, specifically hydroxyl and superoxide radicals, and because of these observations it has been suggested that MT may provide protection against radiation-induced oxidative stress in vivo. Cell lines expressing increased levels of MT have demonstrated resistance to ionizing radiation, to ultraviolet radiation, and also to various DNA damaging agents including melphalan and cis-diaminedichloroplatinum. It is therefore important to gain some insight into the relationship between cellular MT content and cellular resistance to radiation and other DNA damaging agents. In this study we investigated the role of MT in providing protection against monochromatic 365-nm UVA radiation, which is known to generate intracellular reactive oxygen species that are involved in both DNA damage and cell killing. For this purpose, we used zinc acetate, a potent inducer of MT, to elevate MT levels in V79 Chinese hamster fibroblasts prior to UVA exposure and determined cell survival for uninduced and induced cultures. In order to eliminate any zinc effects other than MT induction, we also isolated and characterized cadmium chloride-resistant clones of V79 cells that have increased steady-state levels of both MT mRNA and protein, and we examined their survival characteristics against 365-nm radiation in the absence of zinc acetate. 14 refs., 3 figs.

  8. Fluoxetine Increases the Expression of miR-572 and miR-663a in Human Neuroblastoma Cell Lines

    PubMed Central

    Mundalil Vasu, Mahesh; Anitha, Ayyappan; Takahashi, Taro; Thanseem, Ismail; Iwata, Keiko; Asakawa, Tetsuya; Suzuki, Katsuaki

    2016-01-01

    Evidence suggests neuroprotective effects of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on the developed neurons in the adult brain. In contrast, the drug may be deleterious to immature or undifferentiated neural cells, although the mechanism is unclear. Recent investigations have suggested that microRNAs (miRNA) may be critical for effectiveness of psychotropic drugs including SSRI. We investigated whether fluoxetine could modulate expressions of neurologically relevant miRNAs in two neuroblastoma SK-N-SH and SH-SY5Y cell lines. Initial screening results revealed that three (miR-489, miR-572 and miR-663a) and four (miR-320a, miR-489, miR-572 and miR-663a) miRNAs were up-regulated in SK-N-SH cells and SH-SY5Y cells, respectively, after 24 hours treatment of fluoxetine (1–25 μM). Cell viability was reduced according to the dose of fluoxetine. The upregulation of miR-572 and miR-663a was consistent in both the SH-SY5Y and SK-N-SH cells, confirmed by a larger scale culture condition. Our data is the first in vitro evidence that fluoxetine could increase the expression of miRNAs in undifferentiated neural cells, and that putative target genes of those miRNAs have been shown to be involved in fundamental neurodevelopmental processes. PMID:27716787

  9. Increased expression and copy number amplification of LINE-1 and SINE B1 retrotransposable elements in murine mammary carcinoma progression.

    PubMed

    Gualtieri, Alberto; Andreola, Federica; Sciamanna, Ilaria; Sinibaldi-Vallebona, Paola; Serafino, Annalucia; Spadafora, Corrado

    2013-11-01

    In higher eukaryotic genomes, Long Interspersed Nuclear Element 1 (LINE-1) retrotransposons and endogenous retroviruses represent large families of repeated elements encoding reverse transcriptase (RT) proteins. Short Interspersed Nuclear Element B1 (SINE B1) retrotrasposons do not encode RT, but use LINE-1-derived RT for their retrotransposition. We previously showed that many cancer types have an abundant endogenous RT activity. Inhibition of that activity, by either RNA interference-dependent silencing of active LINE-1 elements or by RT inhibitory drugs, reduced proliferation and promoted differentiation in cancer cells, indicating that LINE-1-encoded RT is required for tumor progression. Using MMTV-PyVT transgenic mice as a well-defined model of breast cancer progression, we now report that both LINE-1 and SINE B1 retrotransposons are up-regulated at a very early stage of tumorigenesis; LINE-1-encoded RT product and enzymatic activity were detected in tumor tissues as early as stage 1, preceding the widespread appearance of histological alterations and specific cancer markers, and further increased in later progression stages, while neither was present in non-pathological breast tissues. Importantly, both LINE-1 and SINE B1 retrotransposon families undergo copy number amplification during tumor progression. These findings therefore indicate that RT activity is distinctive of breast cancer cells and that, furthermore, LINE-1 and SINE B1 undergo copy number amplification during cancer progression.

  10. Establishment and evaluation of a murine αvβ3-integrin-expressing cell line with increased susceptibility to Foot-and-mouth disease virus

    PubMed Central

    Zhang, Wei; Lian, Kaiqi; Yang, Fan; Yang, Yang; Zhu, Zhijian; Zhu, Zixiang; Cao, Weijun; Mao, Ruoqing; Jin, Ye; He, Jijun; Guo, Jianhong; Liu, Xiangtao

    2015-01-01

    Integrin αvβ3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of αvβ3 integrin, a stable CHO-677 cell line expressing the murine αvβ3 heterodimer (designated as "CHO-677-mαvβ3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits αv and β3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-mαvβ3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-mαvβ3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable αvβ3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of αvβ3 integrin, and as a cell model for FMDV research. PMID:25643796

  11. Urbanization increases left-bias in line-bisection: an expression of elevated levels of intrinsic alertness?

    PubMed Central

    Linnell, Karina J.; Caparos, Serge; Davidoff, Jules

    2014-01-01

    Urbanization impairs attentional selection and increases distraction from task-irrelevant contextual information, consistent with a reduction in attentional engagement with the task in hand. Previously, we proposed an attentional-state account of these findings, suggesting that urbanization increases intrinsic alertness and with it exploration of the wider environment at the cost of engagement with the task in hand. Here, we compare urbanized people with a remote people on a line-bisection paradigm. We show that urbanized people have a left spatial bias where remote people have no significant bias. These findings are consistent with the alertness account and provide the first test of why remote peoples have such an extraordinary capacity to concentrate. PMID:25346707

  12. Increased sialyl Lewis A expression and fucosyltransferase activity with acquisition of a high metastatic capacity in a colon cancer cell line.

    PubMed

    Yamada, N; Chung, Y S; Takatsuka, S; Arimoto, Y; Sawada, T; Dohi, T; Sowa, M

    1997-01-01

    A human colon cancer cell line, OCUC-LM1(LM), was established from a liver metastasis in our laboratory. Intrasplenic injection of LM into nude mice was repeated three and five times, and the daughter cell lines were designated as LM-H3 and LM-H5 respectively. The level of sialyl Lewis A (SLA) in the supernatant of LM-H3 and LM-H5 was 3 and 4.5 times higher than that of LM respectively. Flow cytometric analysis of SLA expression showed that the peak channel for LM was 113; for LM-H3, 126; and for LM-H5, 146. The mean fluorescence intensity of LM was 102.3 +/- 43.5; for LM-H3, 126.2 +/- 28.4; and for LM-H5, 144.8 +/- 23.4. In endothelial cell adhesion assays, the percentages of adherent LM-H3 and LM-H5 cells were significantly higher than for LM. The activity of alpha1-->4 fucosyltransferase was higher in LM-H3 and LM-H5 than in LM, but there was no difference in alpha2-->3 sialyltransferase activities for type 1 chain among the cell lines. Our results suggest that SLA expression is associated with acquisition of a high capacity for liver metastasis of colon cancer; increased SLA expression is due mainly to increased fucosyltransferase activity.

  13. Higher Levels of c-Met Expression and Phosphorylation Identify Cell Lines With Increased Sensitivity to AMG-458, a Novel Selective c-Met Inhibitor With Radiosensitizing Effects

    SciTech Connect

    Li Bo; Torossian, Artour; Sun, Yunguang; Du, Ruihong; Dicker, Adam P.; Lu Bo

    2012-11-15

    Purpose: c-Met is overexpressed in some non-small cell lung cancer (NSCLC) cell lines and tissues. Cell lines with higher levels of c-Met expression and phosphorylation depend on this receptor for survival. We studied the effects of AMG-458 on 2 NSCLC cell lines. Methods and Materials: 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium assays assessed the sensitivities of the cells to AMG-458. Clonogenic survival assays illustrated the radiosensitizing effects of AMG-458. Western blot for cleaved caspase 3 measured apoptosis. Immunoblotting for c-Met, phospho-Met (p-Met), Akt/p-Akt, and Erk/p-Erk was performed to observe downstream signaling. Results: AMG-458 enhanced radiosensitivity in H441 but not in A549. H441 showed constitutive phosphorylation of c-Met. A549 expressed low levels of c-Met, which were phosphorylated only in the presence of exogenous hepatocyte growth factor. The combination of radiation therapy and AMG-458 treatment was found to synergistically increase apoptosis in the H441 cell line but not in A549. Radiation therapy, AMG-458, and combination treatment were found to reduce p-Akt and p-Erk levels in H441 but not in A549. H441 became less sensitive to AMG-458 after small interfering RNA knockdown of c-Met; there was no change in A549. After overexpression of c-Met, A549 became more sensitive, while H441 became less sensitive to AMG-458. Conclusions: AMG-458 was more effective in cells that expressed higher levels of c-Met/p-Met, suggesting that higher levels of c-Met and p-Met in NSCLC tissue may classify a subset of tumors that are more sensitive to molecular therapies against this receptor.

  14. Increased expression of the MGMT repair protein mediated by cysteine prodrugs and chemopreventative natural products in human lymphocytes and tumor cell lines.

    PubMed

    Niture, Suryakant K; Velu, Chinavenmani S; Smith, Quentin R; Bhat, G Jayarama; Srivenugopal, Kalkunte S

    2007-02-01

    O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein which protects the cellular genome and critical oncogenic genes from the mutagenic action of endogenous and exogenous alkylating agents. An expedited elimination of O6-alkylguanines by increasing MGMT activity levels is likely to be a successful chemoprevention strategy. Here, we report for the first time that cysteine/glutathione enhancing drugs and certain plant antioxidants possess the ability to increase human MGMT expression beyond its steady-state levels that may afford protection. The non-toxic cysteine prodrugs, 2-oxothiazolidine-4-carboxylic acid (OTC) and N-acetyl-L-cysteine (NAC), metabolized, respectively by 5-oxoprolinase and acylases, increased the MGMT protein and its repair activity levels in a dose- and time-dependent manner in several cancer cell lines and peripheral blood lymphocytes with a maximum of 3-fold increase by 72 h. The natural antioxidants, namely, curcumin, silymarin, sulforaphane and resveratrol were also effective in raising the MGMT levels to different extents. Among the synthetic agents, oltipraz and N-(4-hydroxyphenyl) retinamide (4-HPR) also increased MGMT expression, albeit to a lesser extent. Augmented mRNA levels accounted at least, in part, for the increased activity of MGMT in this setting. However, evidence from cysteine/methionine deprivation, acivicin treatment, and protein synthesis measurements in OTC-treated cells suggested that an increased cysteine flux also contributed significantly to enhanced MGMT expression. Many of these treatments increased the glutathione S-transferase-P1 (GSTP1) levels as well. These findings raise the possibility of MGMT-targeted chemoprevention strategies through dietary supplementation of OTC and herbal antioxidants. Further, the studies reveal the antioxidant responsiveness of the human MGMT gene. PMID:16950796

  15. Obesity-associated proinflammatory cytokines increase calcium sensing receptor (CaSR) protein expression in primary human adipocytes and LS14 human adipose cell line.

    PubMed

    Cifuentes, Mariana; Fuentes, Cecilia; Mattar, Pamela; Tobar, Nicolas; Hugo, Eric; Ben-Jonathan, Nira; Rojas, Cecilia; Martínez, Jorge

    2010-08-15

    Obesity-associated health complications are thought to be in part due to the low-grade proinflammatory state that characterizes this disease. The calcium sensing receptor (CaSR), which is expressed in human adipose cells, plays an important role in diseases involving inflammation. To assess the relevance of this protein in adipose pathophysiology, we evaluated its expression in adipocytes under obesity-related proinflammatory conditions. As in primary adipose cells, we established that LS14, a recently described human adipose cell line, expresses the CaSR. Differentiated LS14 and primary adipose cells were exposed overnight to cytokines typically involved in obesity-related inflammation (interleukin (IL)1beta, IL6 and tumor necrosis factor (TNF)alpha). The cytokines increased CaSR abundance in differentiated adipocytes. We incubated LS14 cells with medium previously conditioned (CM) by adipose tissue from subjects with a wide range of body mass index (BMI). Cells exposed to CM from subjects of higher BMI underwent a greater increase in CaSR protein, likely resulting from the greater proinflammatory cytokines secreted from obese tissue. Our observations that proinflammatory factors increase CaSR levels in adipocytes, and the reported ability of CaSR to elevate cytokine levels, open new aspects in the study of obesity inflammatory state pathophysiology, providing a potential novel therapeutic prevention and treatment target.

  16. HLA expression in hepatocellular carcinoma cell lines.

    PubMed

    Wadee, A A; Paterson, A; Coplan, K A; Reddy, S G

    1994-08-01

    The present study undertook to investigate the biological significance of human leucocyte antigen expression in hepatocellular carcinoma and to elucidate the role of potential modulating agents on human leucocyte antigen expression. These studies used several hepatic tumour-derived cell lines as in vitro model systems. The cell lines included PLC/PRF/5 (Alexander cell line), Hep3B, HepG2, TONG PHC, HA22T/VGH, HA59T/VGH and Mahlavu. The cell lines K562 and Raji were used as negative and positive controls, respectively. K562, a B lymphoid-derived cell line, was shown to express negligible amounts of human leucocyte antigens, while Raji, an erythromyeloid-derived cell line, expressed both class I and class II human leucocyte antigens as well as their respective invariant chains, beta 2-microglobulin and Ii. Using an ELISA, experiments performed on these cell lines confirmed the natural expression of class I and class II antigens by the HA22T/VGH and HA59T/VGH cell lines, whereas PLC/PRF/5 displayed class II surface antigens only. The effects of modulating agents such as interferon-gamma sodium butyrate and clofazimine on human leucocyte antigen expression were investigated using the HA22T/VGH, HA59T/VGH and TONG PHC cell lines. These agents increased class II and class II human leucocyte antigen expression on HA22T/VGH and TONG PHC cells, but had no effect on the HA59T/VGH cell line. The results suggest a potential use for these agents as modulators of human leucocyte antigen expression by human heptocellular cell lines.

  17. Expression of melanocortin receptors in human prostate cancer cell lines: MC2R activation by ACTH increases prostate cancer cell proliferation.

    PubMed

    Hafiz, Saly; Dennis, John C; Schwartz, Dean; Judd, Robert; Tao, Ya-Xiong; Khazal, Kamel; Akingbemi, Benson; Mo, Xiu-Lei; Abdel-Mageed, Asim B; Morrison, Edward; Mansour, Mahmoud

    2012-10-01

    The melanocortin receptors (MCRs 1-5) are G protein coupled-receptors (GPCRs) that regulate food intake, inflammation, skin pigmentation, sexual function and steroidogenesis. Their peptide ligands, the melanocortins, are α-, β- and γ-melanocyte-stimulating hormone and adrenocorticotropic hormone (ACTH) all of which are secreted from the anterior pituitary gland under hypothalamic control. MC2R binds ACTH but has no affinity for the other melanocortins and is, thereby, pharmacologically different from MCRs that bind those ligands. Evidence suggests that elevated GPCRs transactivate the androgen receptor (AR), the critical mediator of prostate cell growth, and consequently promote prostate cancer cell proliferation. It may be that reduced central melanocortin signaling is coincidental with reversal of prostate cancer cachexia, but no data are available on the expression of, or the role for, MCRs in prostate cancer. Here, we show that MCR (1-5) mRNAs are expressed in androgen-dependent LNCaP and androgen-independent PC3 and DU-145 human prostate cancer cell lines. Further, MC2R, the specific target of ACTH, is expressed in LNCaP, PC3 and DU-145 cells. Among the several synthetic MCR peptide ligands that we used, only ACTH promoted concentration-dependent cell proliferation in the three cell lines as shown by MTT cell proliferation assay. In LNCaP cells, the effect was additive with testosterone stimulation and was partially blunted with SHU9119, a non-selective MCR antagonist. In the same cells, ACTH induced cAMP production and increased AR nuclear labeling in immunocytochemical assays. Our observations suggest that MC2R is involved in prostate carcinogenesis and that targeting MC2R signaling may provide a novel avenue in prostate carcinoma treatment. PMID:22842514

  18. Increased in vitro and in vivo tumoricidal activity of a macrophage cell line genetically engineered to express IFN-gamma, IL-4, IL-6, or TNF-alpha.

    PubMed

    Nishihara, K; Barth, R F; Wilkie, N; Lang, J C; Oda, Y; Kikuchi, H; Everson, M P; Lotze, M T

    1995-06-01

    Genetically engineered monocytes and macrophages may have potential as effector cells for the adoptive immunotherapy of cancer. As a first step, we have transfected the genes encoding either mouse interferon (IFN)-gamma, human interleukin (IL)-6, mouse IL-4, or mouse tumor necrosis factor (TNF)-alpha into the mouse macrophage cell line, J774A.1 cells using retroviral vectors. In vitro activation of J774A.1 cells by gene modification was assessed by morphological changes, proliferative activity was determined by [3H]-TdR uptake, and cytolytic activity was assessed using an 18-hour chromium-51 (51Cr) release assay. In vivo tumoricidal activity was studied by means of local adoptive immunotherapy using intratumoral injection of transfected effector cells. IFN-gamma gene-transfected J774A.1 [J7(IFN-gamma)] cells developed filamentous processes, increased doubling times, and enhanced tumoricidal activity against three tumor cell lines: the TNF-sensitive fibrosarcoma line WEHI 164 and the TNF-alpha-resistant cell lines B16 melanoma and C1300 neuroblastoma. IL-6-, TNF-alpha-, and IL-4-gene-transfected J774A.1 cells also had augmented tumoricidal activity but did not display any changes in morphology or growth. Cytolytic activity was markedly reduced after the addition of anti-TNF-alpha antibodies. Cytolytic J7(IFN-gamma) cells showed upregulated expression of TNF-alpha messenger RNA. After intratumoral injection of J7(IL-4) and J7(IFN-gamma) cell mixtures, 50% of established B16 melanomas were rejected by C57BL/6 mice, thereby demonstrating synergistic killing. Further studies on gene-transfected macrophages should better define their potential usefulness in tumor immunotherapy.

  19. Targeting MAGE-C1/CT7 Expression Increases Cell Sensitivity to the Proteasome Inhibitor Bortezomib in Multiple Myeloma Cell Lines

    PubMed Central

    de Carvalho, Fabricio; Costa, Erico T.; Camargo, Anamaria A.; Gregorio, Juliana C.; Masotti, Cibele; Andrade, Valeria C.C.; Strauss, Bryan E.; Caballero, Otavia L.; Atanackovic, Djordje; Colleoni, Gisele W.B.

    2011-01-01

    The MAGE-C1/CT7 encodes a cancer/testis antigen (CTA), is located on the chromosomal region Xq26–27 and is highly polymorphic in humans. MAGE-C1/CT7 is frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. MAGEC1/CT7 expression is restricted to malignant plasma cells and it has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function this protein in the pathophysiology of MM is not yet understood. Our objectives were (1) to clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle in myeloma and (2) to evaluate the impact of silencing MAGE-C1/CT7 on myeloma cells treated with bortezomib. Myeloma cell line SKO-007 was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time quantitative PCR and western blot. Functional assays included cell proliferation, cell invasion, cell cycle analysis and apoptosis. Western blot showed a 70–80% decrease in MAGE-C1/CT7 protein expression in inhibited cells (shRNA-MAGE-C1/CT7) when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. However, we found a decreased percentage of cells in the G2/M phase of the cell cycle among inhibited cells, but not in the controls (p<0.05). When myeloma cells were treated with bortezomib, we observed a 48% reduction of cells in the G2/M phase among inhibited cells while controls showed 13% (empty vector) and 9% (ineffective shRNA) reduction, respectively (p<0.01). Furthermore, inhibited cells treated with bortezomib showed an increased percentage of apoptotic cells (Annexin V+/PI-) in comparison with bortezomib-treated controls (p<0.001). We found that MAGE-C1/CT7 protects SKO-007 cells against bortezomib-induced apoptosis. Therefore, we could speculate that MAGE-C1/CT7 gene therapy could be

  20. Significantly increased expression of OCT4 and ABCG2 in spheroid body-forming cells of the human gastric cancer MKN-45 cell line.

    PubMed

    Liu, Jianming; Wang, Lei; Ma, Lilin; Xu, Junfei; Liu, Chun; Zhang, Jianguo; Liu, Jie; Chen, Ruixin

    2013-10-01

    The cancer stem cell (CSC) theory hypothesizes that CSCs are the cause of tumor formation, recurrence and metastasis. Key to the study of CSCs is their isolation and identification. The present study investigated whether spheroid body-forming cells in the human gastric cancer (GC) MKN-45 cell line are enriched for CSC properties, and also assessed the expression of the candidate CSC markers, octamer-binding transcription factor-4 (OCT4) and adenosine triphosphate-binding cassette transporter G2 (ABCG2) in the MKN-45 spheroid body cells. The MKN-45 cells were plated in a stem cell-conditioned culture system to allow for spheroid body formation. The expression levels of OCT4 and ABCG2 in the spheroid body cells were assessed by qPCR, western blot analysis and immunofluorescence staining, while the tumorigenicity of the spheroid body-forming cells was assessed by in vivo xenograft studies in nude mice. The MKN-45 cells were able to form spheroid bodies when cultured in stem cell-conditioned medium. The spheroid body-forming cells showed a significantly higher (P<0.01) expression of OCT4 and ABCG2 compared with the parental cells. These data suggest that the spheroid body cells from the MKN-45 GC cell line cultured in stem cell-conditioned medium possessed gastric CSC properties. The co-expression of OCT4 and ABCG2 by these cells may represent the presence of a subpopulation of gastric CSCs.

  1. Androgen Receptor Increases CD133 Expression and Progenitor-Like Population That Associate With Cisplatin Resistance in Endometrial Cancer Cell Line

    PubMed Central

    Chen, Lumin; Chang, Wei-Chun; Hung, Yao-Ching; Chang, Ying-Yi; Bao, Bo-Yin; Huang, Hsin-Ching; Chung, Wei-Min; Shyr, Chih-Rong

    2014-01-01

    Endometrial cancer (EMC) is a sex steroid hormone-related female malignancy. Androgen and androgen receptor (androgen/AR) signals have been implicated in EMC progression. Cancer stem/progenitor cells (CSPCs) are suspected to link to chemoresistance in patients with EMC. In this study, we examined the androgen/AR roles in cisplatin resistance and CSPC population. We found AR expression increased naive EMC side population, CSPC population, cell migration, and epithelial–mesenchymal transition. Meanwhile, it decreased cisplatin cytotoxic effect on EMC cells. Collaterally, endogenous AR expressions in EMC cells were upregulated in the cisplatin-resisting state. Moreover, AR expression could further enhance CD133 expression, CSPC-related markers, and drug-resistance gene messenger RNA expression in EMC cells. Finally, the AR-associated gene expression might go through indirect regulation. This is the first report revealing AR function on EMC cells’ CSPC and cisplatin resistance. PMID:23962788

  2. Therapeutic concentrations of valproate but not amitriptyline increase neuropeptide Y (NPY) expression in the human SH-SY5Y neuroblastoma cell line.

    PubMed

    Farrelly, Lorna A; Savage, Niall T P; O'Callaghan, Cristina; Toulouse, André; Yilmazer-Hanke, Deniz M

    2013-09-10

    Neuropeptide Y (NPY) is a peptide found in the brain and autonomic nervous system, which is associated with anxiety, depression, epilepsy, learning and memory, sleep, obesity and circadian rhythms. NPY has recently gained much attention as an endogenous antiepileptic and antidepressant agent, as drugs with antiepileptic and/or mood-stabilizing properties may exert their action by increasing NPY concentrations, which in turn can reduce anxiety and depression levels, dampen seizures or increase seizure threshold. We have used human neuroblastoma SH-SY5Y cells to investigate the effect of valproate (VPA) and amitriptyline (AMI) on NPY expression at therapeutic plasma concentrations of 0.6mM and 630nM, respectively. In addition, 12-O-tetradecanoylphorbol-13-acetate (TPA) known to differentiate SH-SY5Y cells into a neuronal phenotype and to increase NPY expression through activation of protein kinase C (PKC) was applied as a positive control (16nM). Cell viability after drug treatment was tested with a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. NPY expression was measured using immunofluorescence and quantitative RT-PCR (qRT-PCR). Results from immunocytochemistry have shown NPY levels to be significantly increased following a 72h but not 24h VPA treatment. A further increase in expression was observed with simultaneous VPA and TPA treatment, suggesting that the two agents may increase NPY expression through different mechanisms. The increase in NPY mRNA by VPA and TPA was confirmed with qRT-PCR after 72h. In contrast, AMI had no effect on NPY expression in SH-SY5Y cells. Together, the data point to an elevation of human NPY mRNA and peptide levels by therapeutic concentrations of VPA following chronic treatment. Thus, upregulation of NPY may have an impact in anti-cancer treatment of neuroblastomas with VPA, and antagonizing hypothalamic NPY effects may help to ameliorate VPA-induced weight gain and obesity without interfering with the

  3. Increasing Originality in Written Expression.

    ERIC Educational Resources Information Center

    Belasco, Jack Thomas

    This study partially replicated Moss's "A Study of the Effect of Selected Methods of Instruction Designed to Increase Originality in Written Expression," except for the fact that this investigator taught a 5th grade and an 11th grade class for most of a school year. Some of the conclusions of the study were: no particular teaching technique was…

  4. Overexpression of tissue-nonspecific alkaline phosphatase increases the expression of neurogenic differentiation markers in the human SH-SY5Y neuroblastoma cell line.

    PubMed

    Graser, Stephanie; Mentrup, Birgit; Schneider, Doris; Klein-Hitpass, Ludger; Jakob, Franz; Hofmann, Christine

    2015-10-01

    Patients suffering from the rare hereditary disease hypophosphatasia (HPP), which is based on mutations in the ALPL gene, tend to develop central nervous system (CNS) related issues like epileptic seizures and neuropsychiatric illnesses such as anxiety and depression, in addition to well-known problems with the mineralization of bones and teeth. Analyses of the molecular role of tissue-nonspecific alkaline phosphatase (TNAP) in transgenic SH-SY5Y(TNAPhigh) neuroblastoma cells compared to SH-SY5Y(TNAPlow) cells indicate that the enzyme influences the expression levels of neuronal marker genes like RNA-binding protein, fox-1 homolog 3 (NEUN) and enolase 2, gamma neuronal (NSE) as well as microtubule-binding proteins like microtubule-associated protein 2 (MAP2) and microtubule-associated protein tau (TAU) during neurogenic differentiation. Fluorescence staining of SH-SY5Y(TNAPhigh) cells reveals TNAP localization throughout the whole length of the developed projection network and even synapsin Ι co-localization with strong TNAP signals at some spots at least at the early time points of differentiation. Additional immunocytochemical staining shows higher MAP2 expression in SH-SY5Y(TNAPhigh) cells and further a distinct up-regulation of tau and MAP2 in the course of neurogenic differentiation. Interestingly, transgenic SH-SY5Y(TNAPhigh) cells are able to develop longer cellular processes compared to control cells after stimulation with all-trans retinoic acid (RA). Current therapies for HPP prioritize improvement of the bone phenotype. Unraveling the molecular role of TNAP in extraosseous tissues, like in the CNS, will help to improve treatment strategies for HPP patients. Taking this rare disease as a model may also help to dissect TNAP's role in neurodegenerative diseases and even improve future treatment of common pathologies.

  5. Expression of human cell cycle regulators in the primary cell line of the African savannah elephant (loxodonta africana) increases proliferation until senescence, but does not induce immortalization.

    PubMed

    Fukuda, Tomokazu; Iino, Yuuka; Onuma, Manabu; Gen, Bando; Inoue-Murayama, Miho; Kiyono, Tohru

    2016-01-01

    The African savannah elephant (Loxodonta africana) is one of the critically endangered animals. Conservation of genetic and cellular resources is important for the promotion of wild life-related research. Although primary cultured cells are a useful model for the physiology and genomics of the wild-type animals, their distribution is restricted due to the limited number of cell divisions allowed in them. Here, we tried to immortalize a primary cell line of L. africana with by overexpressing human mutant form of cyclin-dependent kinase 4 (CDK4R24C), cyclin D, and telomerase (TERT). It has been shown before that the combination of human CDK4R24C, cyclin D, and TERT induces the efficient cellular immortalization of cells derived from humans, bovine, swine, and monkeys. Interestingly, although the combination of these three genes extended the cellular proliferation of the L. africana-derived cells, they did not induce cellular immortalization. This study suggest that control of cellular senescence in L. africana-derived cells would be different molecular mechanisms compared to those governing human, bovine, swine, and monkey cells.

  6. Complement-dependent cellular cytotoxicity: lymphoblastoid lines that activate complement component 3 (C3) and express C3 receptors have increased sensitivity to lymphocyte-mediated lysis in the presence of fresh human serum.

    PubMed

    Ramos, O F; Sármay, G; Klein, E; Yefenof, E; Gergely, J

    1985-08-01

    Lymphocyte-mediated lysis of cells of the Raji, Daudi, Jijoye, and Bjab lines was elevated when fresh human serum was added to the assay. A higher proportion of effector-target conjugates was observed in the presence of human serum. In similar experiments lysis of 1301, Rael, and P3HR-1 cells was unaltered. All cell lines activated the alternative pathway of complement but they varied in the expression of receptors for complement component 3 (C3) and in the ability to fix the C3 cleavage products on their membrane. The enhancement of lysis in the presence of human serum occurred only with those cells that bound C3. This characteristic was correlated to the expression of C3 receptors. Analysis of the nature of the deposited C3 was performed with Raji cells. Raji cells exposed to human serum bound C3b as indicated by the immunoadherence test. The C3b was further processed to C3bi, because the immunoadherence declined with time and conjugate formation increased with Daudi cells, which carry the C3 receptors CR2 and CR3. This suggests that in the lytic assay lymphocytes with C3bi receptors are recruited in the presence of human serum. We assume that the bridge of C3 molecules between targets and effectors increases the avidity of their interaction. PMID:3895232

  7. Somatic expression of LINE-1 elements in human tissues.

    PubMed

    Belancio, Victoria P; Roy-Engel, Astrid M; Pochampally, Radhika R; Deininger, Prescott

    2010-07-01

    LINE-1 expression damages host DNA via insertions and endonuclease-dependent DNA double-strand breaks (DSBs) that are highly toxic and mutagenic. The predominant tissue of LINE-1 expression has been considered to be the germ line. We show that both full-length and processed L1 transcripts are widespread in human somatic tissues and transformed cells, with significant variation in both L1 expression and L1 mRNA processing. This is the first demonstration that RNA processing is a major regulator of L1 activity. Many tissues also produce translatable spliced transcript (SpORF2). An Alu retrotransposition assay, COMET assays and 53BP1 foci staining show that the SpORF2 product can support functional ORF2 protein expression and can induce DNA damage in normal cells. Tests of the senescence-associated beta-galactosidase expression suggest that expression of exogenous full-length L1, or the SpORF2 mRNA alone in human fibroblasts and adult stem cells triggers a senescence-like phenotype, which is one of the reported responses to DNA damage. In contrast to previous assumptions that L1 expression is germ line specific, the increased spectrum of tissues exposed to L1-associated damage suggests a role for L1 as an endogenous mutagen in somatic tissues. These findings have potential consequences for the whole organism in the form of cancer and mammalian aging.

  8. Regulated expression of erythropoietin by two human hepatoma cell lines

    SciTech Connect

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  9. Lead exposure increases blood pressure by increasing angiotensinogen expression.

    PubMed

    Jiao, Jiandong; Wang, Miaomiao; Wang, Yiqing; Sun, Na; Li, Chunping

    2016-01-01

    Lead exposure can induce increased blood pressure. Several mechanisms have been proposed to explain lead-induced hypertension. Changes in angiotensinogen (AGT) expression levels or gene variants may also influence blood pressure. In this study, we hypothesized that AGT expression levels or gene variants contribute to lead-induced hypertension. A preliminary HEK293 cell model experiment was performed to analyze the association between AGT expression and lead exposure. In a population-based study, serum AGT level was measured in both lead-exposed and control populations. To further detect the influence of AGT gene single nucleotide polymorphisms (SNPs) in lead-induced hypertension, two SNPs (rs699 and rs4762) were genotyped in a case-control study including 219 lead-exposed subjects and 393 controls. Lead exposure caused an increase in AGT expression level in HEK 293 cell models (P < 0.001) compared to lead-free cells, and individuals exposed to lead had higher systolic and diastolic blood pressure (P < 0.001). Lead-exposed individuals had higher serum AGT levels compared to controls (P < 0.001). However, no association was found between AGT gene SNPs (rs699 and rs4762) and lead exposure. Nevertheless, the change in AGT expression level may play an important role in the development of lead-induced hypertension.

  10. Red Maca (Lepidium meyenii) did not affect cell viability despite increased androgen receptor and prostate-specific antigen gene expression in the human prostate cancer cell line LNCaP.

    PubMed

    Díaz, P; Cardenas, H; Orihuela, P A

    2016-10-01

    We examined whether aqueous extract of Lepidium meyenii (red Maca) could inhibit growth, potentiate apoptotic activity of two anticancer drugs Taxol and 2-methoxyestradiol (2ME) or change mRNA expression for the androgen target genes, androgen receptor (Ar) and prostate-specific antigen (Psa) in the human prostate cancer cell line LNCaP. Red Maca aqueous extract at 0, 10, 20, 40 or 80 μg/ml was added to LNCaP cells, and viability was evaluated by the MTS assay at 24 or 48 hr after treatment. Furthermore, LNCaP cells were treated with 80 μg/ml of red Maca plus Taxol or 2ME 5 μM and viability was assessed 48 hr later. Finally, LNCaP cells were treated with red Maca 0, 20, 40 or 80 μg/ml, and 12 hr later, mRNA level for Ar or Psa was assessed by real-time PCR. Treatment with red Maca did not affect viability of LNCaP cells. Apoptotic activity induced by Taxol and 2ME in LNCaP cells was not altered with red Maca treatment. Relative expression of the mRNA for Ar and Psa increased with red Maca 20 and 40 μg/ml, but not at 80 μg/ml. We conclude that red Maca aqueous extract does not have toxic effects, but stimulates androgen signalling in LNCaP cells. PMID:27681649

  11. Red Maca (Lepidium meyenii) did not affect cell viability despite increased androgen receptor and prostate-specific antigen gene expression in the human prostate cancer cell line LNCaP.

    PubMed

    Díaz, P; Cardenas, H; Orihuela, P A

    2016-10-01

    We examined whether aqueous extract of Lepidium meyenii (red Maca) could inhibit growth, potentiate apoptotic activity of two anticancer drugs Taxol and 2-methoxyestradiol (2ME) or change mRNA expression for the androgen target genes, androgen receptor (Ar) and prostate-specific antigen (Psa) in the human prostate cancer cell line LNCaP. Red Maca aqueous extract at 0, 10, 20, 40 or 80 μg/ml was added to LNCaP cells, and viability was evaluated by the MTS assay at 24 or 48 hr after treatment. Furthermore, LNCaP cells were treated with 80 μg/ml of red Maca plus Taxol or 2ME 5 μM and viability was assessed 48 hr later. Finally, LNCaP cells were treated with red Maca 0, 20, 40 or 80 μg/ml, and 12 hr later, mRNA level for Ar or Psa was assessed by real-time PCR. Treatment with red Maca did not affect viability of LNCaP cells. Apoptotic activity induced by Taxol and 2ME in LNCaP cells was not altered with red Maca treatment. Relative expression of the mRNA for Ar and Psa increased with red Maca 20 and 40 μg/ml, but not at 80 μg/ml. We conclude that red Maca aqueous extract does not have toxic effects, but stimulates androgen signalling in LNCaP cells.

  12. Lead induces increased water permeability in astrocytes expressing aquaporin 4.

    PubMed

    Gunnarson, E; Axehult, G; Baturina, G; Zelenin, S; Zelenina, M; Aperia, A

    2005-01-01

    The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes. There is now compelling evidence that AQP4 may contribute to an unfavorable course in brain edema. Acute lead intoxication is a condition that causes brain damage preceded by brain edema. Here we report that lead increases AQP4 water permeability (P(f)) in astrocytes. A rat astrocyte cell line that does not express aquaporin 4 was transiently transfected with aquaporin 4 tagged with green fluorescent protein (GFP). Using confocal laser scanning microscopy we measured water permeability in these cells and in AQP4-negative cells located on the same plate. AQP4-expressing astrocytes had a three-fold higher water permeability than astrocytes not expressing AQP4. Lead exposure induced a significant, 40%, increase in water permeability in astrocytes expressing AQP4, but had no effect on P(f) in astrocytes not expressing AQP4. The increase in water permeability persisted after lead washout, while treatment with a lead chelator, meso-2,3-dimercaptosuccinic acid, abolished the lead-induced increase in P(f). The effect of lead was attenuated in the presence of a calcium (Ca(2+))/calmodulin-dependent protein kinase II (CaMKII) inhibitor, but not in the presence of a protein kinase C inhibitor. In cells expressing AQP4 where the consensus site for CaMKII phosphorylation was mutated, lead failed to increase water permeability. Lead exposure also increased P(f) in rat astroglial cells in primary culture, which express endogenous AQP4. Lead had no effect on P(f) in astrocytes transfected with aquaporin 3. In situ hybridization studies on rat brain after oral lead intake for three days showed no change in distribution of AQP4 mRNA. It is suggested that lead-triggered stimulation of water transport in AQP4-expressing astrocytes may contribute to the pathology of acute lead intoxication.

  13. Expression of Tropomyosin 1 Gene Isoforms in Human Breast Cancer Cell Lines

    PubMed Central

    Dube, Syamalima; Yalamanchili, Santhi; Lachant, Joseph; Abbott, Lynn; Benz, Patricia; Mitschow, Charles; Dube, Dipak K.; Poiesz, Bernard J.

    2015-01-01

    Nine malignant breast epithelial cell lines and 3 normal breast cell lines were examined for stress fiber formation and expression of TPM1 isoform-specific RNAs and proteins. Stress fiber formation was strong (++++) in the normal cell lines and varied among the malignant cell lines (negative to +++). Although TPM1γ and TPM1δ were the dominant transcripts of TPM1, there was no clear evidence for TPM1δ protein expression. Four novel human TPM1 gene RNA isoforms were discovered (λ, μ, ν, and ξ), which were not identified in adult and fetal human cardiac tissues. TPM1λ was the most frequent isoform expressed in the malignant breast cell lines, and it was absent in normal breast epithelial cell lines. By western blotting, we were unable to distinguish between TPM1γ, λ, and ν protein expression, which were the only TPM1 gene protein isoforms potentially expressed. Some malignant cell lines demonstrated increased or decreased expression of these isoforms relative to the normal breast cell lines. Stress fiber formation did not correlate with TPM1γ RNA expression but significantly and inversely correlated with TPM1δ and TPM1λ expression, respectively. The exact differences in expression of these novel isoforms and their functional properties in breast epithelial cells will require further study. PMID:26171250

  14. The increases in mRNA expressions of inflammatory cytokines by adding cleaning solvent or tetrachloroethylene in the murine macrophage cell line J774.1 evaluated by real-time PCR.

    PubMed

    Kido, Takamasa; Sugaya, Chiemi; Ikeuchi, Ryutaro; Kudo, Yuichiro; Tsunoda, Masashi; Aizawa, Yoshiharu

    2013-01-01

    The use of a petroleum-derived cleaning solvent for dry cleaning, instead of tetrachloroethylene (perchloroethylene, PCE), has increased. The cleaning solvent may induce immunological alteration. In this study, murine macrophage-lineage J774.1 cells were exposed to the cleaning solvent at 0, 25, 50, and 75 µg/ml or PCE at 0, 400, 600, 800, and 1,000 µg/ml by vigorous vortexing. Cell viability was determined. The mRNA expressions of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, IL-10, IL-12p40 (a dimer of IL-12), and IL-27p28 (a dimer of IL-27) were evaluated by real-time PCR. The mean viabilities in the 50 and 75 µg/ml groups of the cleaning solvent were significantly lower than that of the control. The mean mRNA expressions of TNF-α and IL-1β in the 50 µg/ml group were significantly higher than those in the control. For PCE, the mean viabilities at 600 µg/ml and over were significantly lower than that of the control. The mean expressions of IL-6 and IL-10 in the 800 µg/ml group were significantly higher than that in the control. The productions of IL-1β and TNF-α may be altered in human during intoxication of the cleaning solvent as well as those of IL-6 and IL-10 in human during that of PCE, and these may affect on immune cells.

  15. Expression of MIF and CD74 in leukemic cell lines: correlation to DR expression destiny.

    PubMed

    Georgouli, Mirella; Papadimitriou, Lina; Glymenaki, Maria; Patsaki, Valia; Athanassakis, Irene

    2016-06-01

    Invariant chain (Ii) or CD74 is a non-polymorphic glycoprotein, which apart from its role as a chaperone dedicated to MHCII molecules, is known to be a high-affinity receptor for macrophage migration inhibitory factor (MIF). The present study aimed to define the roles of CD74 and MIF in the immune surveillance escape process. Towards this direction, the cell lines HL-60, Raji, K562 and primary pre-B leukemic cells were examined for expression and secretion of MIF. Flow cytometry analysis detected high levels of MIF and intracellular/membrane CD74 expression in all leukemic cells tested, while MIF secretion was shown to be inversely proportional to intracellular HLA-DR (DR) expression. In the MHCII-negative cells, IFN-γ increased MIF expression and induced its secretion in HL-60 and K562 cells, respectively. In K562 cells, CD74 (Iip33Iip35) was shown to co-precipitate with HLA-DOβ (DOβ), inhibiting thus MIF or DR binding. Induced expression of DOα in K562 (DOα-DOβ+) cells in different transfection combinations decreased MIF expression and secretion, while increasing surface DR expression. Thus, MIF could indeed be part of the antigen presentation process.

  16. Thyroid hormone increases bulk histones expression by enhancing translational efficiency.

    PubMed

    Zambrano, Alberto; García-Carpizo, Verónica; Villamuera, Raquel; Aranda, Ana

    2015-01-01

    The expression of canonical histones is normally coupled to DNA synthesis during the S phase of the cell cycle. Replication-dependent histone mRNAs do not contain a poly(A) tail at their 3' terminus, but instead possess a stem-loop motif, the binding site for the stem-loop binding protein (SLBP), which regulates mRNA processing, stability, and relocation to polysomes. Here we show that the thyroid hormone can increase the levels of canonical histones independent of DNA replication. Incubation of mouse embryonic fibroblasts with T3 increases the total levels of histones, and expression of the thyroid hormone receptor β induces a further increase. This is not restricted to mouse embryonic fibroblasts, because T3 also raises histone expression in other cell lines. T3 does not increase histone mRNA or SLBP levels, suggesting that T3 regulates histone expression by a posttranscriptional mechanism. Indeed, T3 enhanced translational efficiency, inducing relocation of histone mRNA to heavy polysomes. Increased translation was associated with augmented transcription of the eukaryotic translation initiation factor 4 γ2 (EIF4G2). T3 induced EIF4G2 protein and mRNA levels and the thyroid hormone receptor bound to the promoter region of the Eif4g2 gene. Induction of EIF4G2 was essential for T3-dependent histone induction, because depletion of this factor abolished histone increase. These results point out the importance of the thyroid hormones on the posttranscriptional regulation of histone biosynthesis in a cell cycle-independent manner and also suggest the potential regulation of eukaryotic translation by the modulation of the initiation factor EIF4G2, which also operates in the translation of canonical mRNAs.

  17. PD-L1 Expression Is Increased in a Subset of Basal Type Breast Cancer Cells

    PubMed Central

    Soliman, Hatem; Khalil, Farah; Antonia, Scott

    2014-01-01

    Background Tumor cells express programmed death ligand 1 (PD-L1) and is a key immune evasion mechanism. PD-L1 expression in multiple breast cancer cell lines was evaluated to identify intrinsic differences that affect their potential for immune evasion. Methods PD-L1 expression was analyzed in six breast cancer cell lines: AU565&MCF7 (luminal), BT20&HCC1143 (basal A), MDA231&HCC38 (basal B). Surface and intracellular PD-L1 expression +/− interferon γ for 48 hours was measured by flow cytometry. PD-L1 gene expression data for all breast cancer cell lines in the Comprehensive Cell Line Encyclopedia (CCLE) was analyzed. Correlation between PD-L1 levels and clinicopathologic parameters was analyzed within Oncomine datasets. A tissue microarray containing 61 invasive breast cancer primary tumor cores was stained for PD-L1 expression and analyzed. Results Basal breast cancer cells constitutively express the highest levels of PD-L1. All cell lines increased PD-L1 expression with interferon γ, but basal B cells (MDA-231 and HCC38) demonstrated the largest increases. There were no differences in protein localization between cell lines. In the CCLE data, basal cell lines demonstrated higher mean PD-L1 expression compared to luminal cell lines. High PD-L1 expressing basal cell lines over-express genes involved in invasion, proliferation, and chemoresistance compared to low PD-L1 basal cell lines. High PD-L1 basal cell lines had lower expression of IRF2BP2 and higher STAT1 levels compared to low PD-L1 expressing cell lines. Within Oncomine datasets PDL1 mRNA levels were higher in basal type tumors. The TMA analysis demonstrated that lymph node positive cases had higher levels of PD-L1 protein expression compared to lymph node negative cases. Conclusions Basal type breast cancer (especially basal B) express greater levels of PD-L1 constitutively and with IFN γ. High PD-L1 basal cells over-express genes involved in invasion, motility, and chemoresistance. Targeting PD-L1

  18. Increased Cortical Thickness in Professional On-Line Gamers

    PubMed Central

    Hyun, Gi Jung; Shin, Yong Wook; Kim, Bung-Nyun; Cheong, Jae Hoon; Jin, Seong Nam

    2013-01-01

    Objective The bulk of recent studies have tested whether video games change the brain in terms of activity and cortical volume. However, such studies are limited by several factors including cross-sectional comparisons, co-morbidity, and short-term follow-up periods. In the present study, we hypothesized that cognitive flexibility and the volume of brain cortex would be correlated with the career length of on-line pro-gamers. Methods High-resolution magnetic resonance scans were acquired in twenty-three pro-gamers recruited from StarCraft pro-game teams. We measured cortical thickness in each individual using FreeSurfer and the cortical thickness was correlated with the career length and the performance of the pro-gamers. Results Career length was positively correlated with cortical thickness in three brain regions: right superior frontal gyrus, right superior parietal gyrus, and right precentral gyrus. Additionally, increased cortical thickness in the prefrontal cortex was correlated with winning rates of the pro-game league. Increased cortical thickness in the prefrontal and parietal cortices was also associated with higher performance of Wisconsin Card Sorting Test. Conclusion Our results suggest that in individuals without pathologic conditions, regular, long-term playing of on-line games is associated with volume changes in the prefrontal and parietal cortices, which are associated with cognitive flexibility. PMID:24474988

  19. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  20. Analysis of LINE-1 expression in human pluripotent cells.

    PubMed

    Muñoz-Lopez, Martin; Garcia-Cañadas, Marta; Macia, Angela; Morell, Santiago; Garcia-Perez, Jose L

    2012-01-01

    Half of the human genome is composed of repeated DNA, and some types are mobile within our genome (transposons and retrotransposons). Despite their abundance, only a small fraction of them are currently active in our genome (Long Interspersed Element-1 (LINE-1), Alu, and SVA elements). LINE-1 or L1 elements are a family of active non-LTR retrotransposons, the ongoing mobilization of which still impacts our genome. As selfish DNA elements, L1 activity is more prominent in early human development, where new insertions would be transmitted to the progeny. Here, we describe the conventional methods aimed to determine the expression level of LINE-1 elements in pluripotent human cells.

  1. Expression of membrane glycoproteins in normal keratinocytes and squamous carcinoma cell lines

    SciTech Connect

    Rayter, Z. ); McIlhinney, R. ); Gusterson, B. )

    1989-08-01

    Con A acceptor glycoproteins were analyzed by 2D-PAGE and {sup 125}I-Con A overlay in three squamous carcinoma cell lines and compared with those in the simian virus (SV40)-transformed keratinocyte cell line SVK-14 and in normal keratinocytes. The majority of the glycoproteins identified by this technique were expressed at similar levels in all of the cells examined, independent of the culture conditions used. A cell surface glycoprotein gp34 was increased in the tumor cells compared with normal keratinocytes and expression varied with the culture density. Another glycoprotein, gp21, was found to be increased in expression in normal keratinocytes and stratified hyperconfluent cultures of squamous carcinoma cell lines. This paper describes the potential of this technique to identify membrane glycoproteins which may be expressed as a function of proliferation or differentiation.

  2. Celecoxib Up Regulates the Expression of Drug Efflux Transporter ABCG2 in Breast Cancer Cell Lines

    PubMed Central

    Kalalinia, Fatemeh; Elahian, Fatemeh; Mosaffa, Fatemeh; Behravan, Javad

    2014-01-01

    Elevated expression of the drug efflux transporter ABCG2 seems to correlate with multidrug resistance of cancer cells. Specific COX-2 inhibitor celecoxib has been shown to enhance the sensitivity of cancer cells to anticancer drugs. To clarify whether ABCG2 inhibition is involved in the sensitizing effect of celecoxib, we investigated whether the expression of ABCG2 in breast cancer cell lines, could be modulated by celecoxib. The expression of the multidrug resistant gene (ABCG2) at mRNA and protein level was detected by real-time quantitative reverse transcription-polymerase chain reaction and flow cytometry analysis, respectively. Among three human breast cancer cell lines ABCG2 and COX-2 were highly expressed in MCF7-MX and MDA-MB-231 cells, respectively. The COX-2 inhibitor celecoxib up-regulated the expression of ABCG2 mRNA in MCF-7 and MCF7-MX cells, which was accompanied by increased ABCG2 protein expression. While celecoxib was able to block the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated increase in COX-2 expression in MDA-MB-231 cells, it increased the expression of ABCG2 up to 4.27 times to the control level at mRNA level and with less intensity at protein level. Our findings provide evidence that celecoxib up-regulates ABCG2 expression in human breast cancer cells and proposed that ABCG2 is not involved in chemosensitizing effects of celecoxib. PMID:25587329

  3. Matrix attachment regions and regulated transcription increase and stabilize transgene expression.

    PubMed

    Abranches, Rita; Shultz, Randall W; Thompson, William F; Allen, George C

    2005-09-01

    Transgene silencing has been shown to be associated with strong promoters, but it is not known whether the propensity for silencing is caused by the level of transcription, or some other property of the promoter. If transcriptional activity fosters silencing, then transgenes with inducible promoters may be less susceptible to silencing. To test this idea, a doxycycline-inducible luciferase transgene was transformed into an NT1 tobacco suspension culture cell line that constitutively expressed the tetracycline repressor. The inducible luciferase gene was flanked by tobacco Rb7 matrix attachment regions (MAR) or spacer control sequences in order to test the effects of MARs in conjunction with regulated transcription. Transformed lines were grown under continuous doxycycline (CI), or delayed doxycycline induction (DI) conditions. Delayed induction resulted in higher luciferase expression initially, but continued growth in the presence of doxycycline resulted in a reduction of expression to levels similar to those found in continuously induced lines. In both DI and CI treatments, the Rb7 MAR significantly reduced the percentage of silenced lines and increased transgene expression levels. These data demonstrate that active transcription increases silencing, especially in the absence of the Rb7 MAR. Importantly, the Rb7 MAR lines showed higher expression levels under both CI and DI conditions and avoided silencing that may occur in the absence of active transcription such as what would be expected as a result of condensed chromatin spreading. PMID:17173639

  4. Duplicate genes increase gene expression diversity within and between species.

    PubMed

    Gu, Zhenglong; Rifkin, Scott A; White, Kevin P; Li, Wen-Hsiung

    2004-06-01

    Using microarray gene expression data from several Drosophila species and strains, we show that duplicated genes, compared with single-copy genes, significantly increase gene expression diversity during development. We show further that duplicate genes tend to cause expression divergences between Drosophila species (or strains) to evolve faster than do single-copy genes. This conclusion is also supported by data from different yeast strains.

  5. Fluorodeoxyglucose uptake in human cancer cell lines is increased by hypoxia

    SciTech Connect

    Clavo, A.C.; Brown, R.S.; Wahl, R.L.

    1995-09-01

    Malignant neoplasms commonly have increased rates of glucose utilization, poor perfusion and areas of low oxygenation. Autoradiographic studies of excised tumors have shown increased FDG uptake in viable cells near necrotic portions of tumor. We evaluated in vitro whether tumor cell FDG uptake increased with hypoxia. The uptake of {sup 3}H-FDG into two human tumor cell lines (HTB 63 melanoma and HTB 77 IP3 ovarian carcinoma) was determined after exposure to differing oxygen atmospheres ranging from 0% to 20% O{sub 2} for varying time periods. Glucose transport was independently determined as well as estimates of the level of Glut-1 glucose transporter membrane protein. FDG uptake in both the melanoma and the ovarian carcinoma cell lines increased significantly (39.6% {plus_minus} 6.7% and 36.7% {plus_minus}9%, respectively) over basal (20% O{sub 2}) conditions when cells were exposed to a mild hypoxic environment (5% O{sub 2}) for 1.5 hr. With a 4-hr exposure to 1.5% O{sub 2}, the increase in FDG uptake was greater at 52.3% {plus_minus} 8.9% and 43.5% {plus_minus} 19%, respectively. With 4 hr of anoxia, the increase in FDG uptake over basal conditions was 42.7% {plus_minus} 10% and 63.3% {plus_minus} 13.7% for melanoma and ovarian carcinoma cells, respectively. Membrane transport of 3-O-methylglucose (3-OMG) was increased by hypoxia for melanoma and ovarian carcinoma. Immunochemical assays for Glut-1 showed an increase in the membrane expression of the Glut-1 transporter in cells exposed to hypoxia. Hypoxia increases cellular uptake of FDG in two different malignant human cell lines. Increased glucose transport, in part due to increased membrane expression of the Glut-1 glucose transporter, contributes to this phenomenon. Increased FDG uptake in tumors visualized during PET imaging may be partly reflective of tumor hypoxia. 47 refs., 6 figs., 2 tabs.

  6. Regulation of alkaline phosphatase expression in human choriocarcinoma cell lines.

    PubMed Central

    Hamilton, T A; Tin, A W; Sussman, H H

    1979-01-01

    The coincident expression of two structurally distinct isoenzymes of human alkaline phosphatase was demonstrated in two independently derived gestational choriocarcinoma cell lines. These proteins were shown to have enzymatic, antigenic, and physical-chemical properties resembling those of isoenzymes from term placenta and adult liver. The regulation of these isoenzymes has been studied during the exposure of both cell lines to 5-bromodeoxyuridine and dibutyryl cyclic AMP. The responses of the alkaline phosphatase isoenzymes to these agents have also been compared with the response of another protein phenotypic to placenta, the alpha subunit of chorionic gonadotropin. The results show that (i) the separate structural genes coding for placental and liver alkaline phosphatases are regulated in a noncoordinate fashion; (ii) both alkaline phosphatase genes respond independently of the alpha subunit; and (iii) the induction of the placental type isoenzyme occurs via at least two independent pathways. Images PMID:218197

  7. Transition of LINE-1 DNA methylation status and altered expression in first and third trimester placentas.

    PubMed

    He, Zhi-ming; Li, Jinping; Hwa, Yi Lisa; Brost, Brian; Fang, Qun; Jiang, Shi-Wen

    2014-01-01

    DNA methylation plays a critical role in the regulation of gene expression, genomic DNA stability, cell proliferation, and malignant transformation. Common cellular features including fast tissue expansion, invasive growth, and active angiogenesis, have been noticed between placental development and tumorigenesis by many investigators. While the DNA hypomethylation and transcriptional activation of LINE-1 has been found to be a feature of tumorigenesis, it is not clear if similar changes could be involved in placental development. In this study, we assessed LINE-1 methylation in human placentas from different gestational ages and observed a significant decrease of LINE-1 methylation levels in third trimester placentas compared to first trimester placentas. Accompanying with this change is the significantly increased LINE-1 mRNA levels in third trimester placentas. Since no global DNA methylation change was detected between first and third trimesters, LINE-1 methylation changes appeared to be a specific epigenetic entity contributing to placental development. Indeed, further analyses showed that LINE-1 upregulation was correlated with higher levels of PCNA, suggesting a link between LINE-1 activation and fast proliferation of certain cellular components in third trimester placentas. Measurement of the DNMT1, DNMT3A, and DNMT3B expression found a significant reduction of DNMT3B between third and first trimesters, pointing to the possible involvement of this enzyme in the regulation of LINE-1 methylation. Taken together these results provided evidence for a dynamic temporal regulation of LINE-1 methylation and activation during placental development. These studies have laid a foundation for future investigation on the function of LINE-1 expression in human placenta under different patho-physiological conditions.

  8. Transition of LINE-1 DNA Methylation Status and Altered Expression in First and Third Trimester Placentas

    PubMed Central

    Hwa, Yi Lisa; Brost, Brian; Fang, Qun; Jiang, Shi-Wen

    2014-01-01

    DNA methylation plays a critical role in the regulation of gene expression, genomic DNA stability, cell proliferation, and malignant transformation. Common cellular features including fast tissue expansion, invasive growth, and active angiogenesis, have been noticed between placental development and tumorigenesis by many investigators. While the DNA hypomethylation and transcriptional activation of LINE-1 has been found to be a feature of tumorigenesis, it is not clear if similar changes could be involved in placental development. In this study, we assessed LINE-1 methylation in human placentas from different gestational ages and observed a significant decrease of LINE-1 methylation levels in third trimester placentas compared to first trimester placentas. Accompanying with this change is the significantly increased LINE-1 mRNA levels in third trimester placentas. Since no global DNA methylation change was detected between first and third trimesters, LINE-1 methylation changes appeared to be a specific epigenetic entity contributing to placental development. Indeed, further analyses showed that LINE-1 upregulation was correlated with higher levels of PCNA, suggesting a link between LINE-1 activation and fast proliferation of certain cellular components in third trimester placentas. Measurement of the DNMT1, DNMT3A, and DNMT3B expression found a significant reduction of DNMT3B between third and first trimesters, pointing to the possible involvement of this enzyme in the regulation of LINE-1 methylation. Taken together these results provided evidence for a dynamic temporal regulation of LINE-1 methylation and activation during placental development. These studies have laid a foundation for future investigation on the function of LINE-1 expression in human placenta under different patho-physiological conditions. PMID:24821186

  9. Expression of tropomyosin 2 gene isoforms in human breast cancer cell lines

    PubMed Central

    DUBE, SYAMALIMA; THOMAS, ANISH; ABBOTT, LYNN; BENZ, PATRICIA; MITSCHOW, CHARLES; DUBE, DIPAK K.; POIESZ, BERNARD J.

    2016-01-01

    In humans, four tropomyosin genes (TPM1, TPM2, TPM3, and TPM4) are known to produce a multitude of isoforms via alternate splicing and/or using alternate promoters. Expression of tropomyosin has been shown to be modulated at both the transcription and the translational levels. Tropomyosins are known to make up some of the stress fibers of human epithelial cells and differences in their expression has been demonstrated in malignant breast epithelial cell lines compared to 'normal' breast cell lines. We have recently reported the expression of four novel TPM1 isoforms (TPM1λ, TPM1µ, TPM1ν, and TPM1ξ) from human malignant tumor breast cell lines that are not expressed in adult and fetal cardiac tissue. Also, we evaluated their expression in relation to the stress fiber formation. In this study, nine malignant breast epithelial cell lines and three 'normal' breast cell lines were examined for stress fiber formation and expression of tropomyosin 2 (TPM2) isoform-specific RNAs and proteins. Stress fiber formation was assessed by immunofluorescence using Leica AF6000 Deconvolution microscope. Stress fiber formation was strong (++++) in the 'normal' cell lines and varied among the malignant cell lines (negative to +++). No new TPM2 gene RNA isoforms were identified, and TPM2β was the most frequently expressed TPM2 RNA and protein isoform. Stress fiber formation positively correlated with TPM2β RNA or protein expression at high, statistically significant degrees. Previously, we had shown that TPM1δ and TPM1λ positively and inversely, respectively, correlated with stress fiber formation. The most powerful predictor of stress fiber formation was the combination of TPM2β RNA, TPM1δ RNA, and the inverse of TPM1λ RNA expression. Our results suggest that the increased expression of TPM1λ and the decreased expression of TPM1δ RNA and TPM2β may lead to decreased stress fiber formation and malignant transformation in human breast epithelial cells. PMID:27108600

  10. Increased expression of IL-16 in inflammatory bowel disease

    PubMed Central

    Seegert, D; Rosenstiel, P; Pfahler, H; Pfefferkorn, P; Nikolaus, S; Schreiber, S

    2001-01-01

    BACKGROUND—Inflammatory bowel disease (IBD) is characterised by infiltration of inflamed mucosal regions with CD4+ T lymphocytes and other mononuclear cells. Interleukin (IL)-16 exerts a strong chemoattractant activity on CD4+ cells. Moreover, IL-16 activates expression and production of proinflammatory cytokines such as IL-1β, IL-6, IL-15, and tumour necrosis factor α (TNF-α) in human monocytes.
AIM—To examine if IL-16 expression is increased in IBD patients compared with healthy controls.
METHODS—Twenty one patients with IBD (10 with ulcerative colitis (UC), 11 with Crohn's disease (CD)), seven disease specificity controls (DSC), and seven healthy controls were studied. Biopsies were taken during colonoscopies and IL-16 mRNA as well as protein expression were investigated by reverse transcriptase-polymerase chain reaction, ELISA, western blot, and immunohistochemistry.
RESULTS—IL-16 mRNA and protein expression in the colonic mucosa of IBD patients were increased twofold compared with healthy controls, DSC, or IBD patients under steroid treatment. Most of the detected IL-16 protein was in its bioactive 17 kDa form and was predominantly expressed in eosinophils. Increased IL-16 expression in UC patients appeared to be mainly restricted to the inflamed regions of the colonic mucosa. Levels of caspase 3, which processes the 68 kDa IL-16 precursor molecule into the biological active 17 kDa form, were not increased.
CONCLUSIONS—Our results provide evidence that IL-16 expression is significantly increased in the inflamed colonic mucosa of IBD patients but not in control individuals, DSC, or patients under steroid treatment. Therefore, upregulation of IL-16 expression seems to be specific for chronic intestinal inflammation and could lead to increased secretion of other proinflammatory cytokines in IBD.


Keywords: interleukin-16; T lymphocytes; eosinophils; Crohn's disease; ulcerative colitis; inflammatory bowel disease PMID:11171821

  11. Moisture sensors 1980 on-line roles increase

    SciTech Connect

    Bailey, S.J.

    1980-09-01

    A review of on- and off-line moisture and humidity sensors covers instruments based on the capacitive principle such as Panametrics' Aquamax II and those developed by Ondyne, Moisture Control Systems, Phys-Chemical Research Corp., Shaw Instruments, and Diversified Engineering; instruments based on the microwave plus gamma and nuclear plus gamma radiation principles such as those by Kay-Ray and Ohmart Corp., respectively; sensors based on the saturated salt principle such as Foxboro's self-heated lithium chloride sensor; Kahn and Co.'s electrolytic hygrometer for monitoring the moisture content of pure gases or gas mixtures in natural gas transmissions; the Ohaus gravimetric instrument; microprocessor-based titrators by Foxboro Analytical and Photovolt Corp.; instruments which sense moving web moisture such as Beckman Instrument's Hygroline System HMP20 and Hy-Cal Engineering's sensor; IR stack gas analyzers by Anarad Inc. and Moisture Systems Corp.; optical hygrometers by EG and G and General Eastern; Panametrics' Model 4000 moisture computer; and Du Pont's standard 560 analyzer and new controller.

  12. Differential hormonal and gene expression dynamics in two inbred sunflower lines with contrasting dormancy level.

    PubMed

    Roselló, Paula L; Vigliocco, Ana E; Andrade, Andrea M; Riera, Natalí V; Calafat, Mario; Molas, María L; Alemano, Sergio G

    2016-05-01

    Seed germination and dormancy are tightly regulated by hormone metabolism and signaling pathway. We investigated the endogenous content of abscisic acid (ABA), its catabolites, and gibberellins (GAs), as well as the expression level of certain ABA and GAs metabolic and signaling genes in embryo of dry and imbibed cypselas of inbred sunflower (Helianthus annuus L., Asteraceae) lines: B123 (dormant) and B91 (non-dormant). Under our experimental conditions, the expression of RGL2 gene might be related to the ABA peak in B123 line at 3 h of imbibition. Indeed, RGL2 transcripts are absent in dry and early embedded cypselas of the non-dormant line B91. ABA increase was accompanied by a significant ABA-Glucosyl ester (ABA-GE) and phaseic acid (PA) (two ABA catabolites) decrease in B123 line (3 h) which indicates that ABA metabolism seems to be more active in this line, and that it would be involved in the imposition and maintenance of sunflower seed dormancy, as it has been reported for many species. Finally, an increase of bioactive GAs (GA1 and GA3) occurs at 12 h of imbibition in both lines after a decrease in ABA content. This study shows the first report about the RGL2 tissue-specific gene expression in sunflower inbred lines with contrasting dormancy level. Furthermore, our results provide evidence that ABA and GAs content and differential expression of metabolism and signaling genes would be interacting in seed dormancy regulation through a mechanism of action related to embryo itself.

  13. Differential hormonal and gene expression dynamics in two inbred sunflower lines with contrasting dormancy level.

    PubMed

    Roselló, Paula L; Vigliocco, Ana E; Andrade, Andrea M; Riera, Natalí V; Calafat, Mario; Molas, María L; Alemano, Sergio G

    2016-05-01

    Seed germination and dormancy are tightly regulated by hormone metabolism and signaling pathway. We investigated the endogenous content of abscisic acid (ABA), its catabolites, and gibberellins (GAs), as well as the expression level of certain ABA and GAs metabolic and signaling genes in embryo of dry and imbibed cypselas of inbred sunflower (Helianthus annuus L., Asteraceae) lines: B123 (dormant) and B91 (non-dormant). Under our experimental conditions, the expression of RGL2 gene might be related to the ABA peak in B123 line at 3 h of imbibition. Indeed, RGL2 transcripts are absent in dry and early embedded cypselas of the non-dormant line B91. ABA increase was accompanied by a significant ABA-Glucosyl ester (ABA-GE) and phaseic acid (PA) (two ABA catabolites) decrease in B123 line (3 h) which indicates that ABA metabolism seems to be more active in this line, and that it would be involved in the imposition and maintenance of sunflower seed dormancy, as it has been reported for many species. Finally, an increase of bioactive GAs (GA1 and GA3) occurs at 12 h of imbibition in both lines after a decrease in ABA content. This study shows the first report about the RGL2 tissue-specific gene expression in sunflower inbred lines with contrasting dormancy level. Furthermore, our results provide evidence that ABA and GAs content and differential expression of metabolism and signaling genes would be interacting in seed dormancy regulation through a mechanism of action related to embryo itself. PMID:26934102

  14. Inhibition of the liver expression of arylalkylamine N-acetyltransferase increases the expression of angiogenic factors in cholangiocytes

    PubMed Central

    Renzi, Anastasia; Mancinelli, Romina; Onori, Paolo; Franchitto, Antonio; Alpini, Gianfranco; Glaser, Shannon

    2014-01-01

    Background and aims Reduction of biliary serotonin N-acetyltransferase (AANAT) expression and melatonin administration/secretion in cholangiocytes increases biliary proliferation and the expression of SR, CFTR and Cl–/HCO3– AE2. The balance between biliary proliferation/damage is regulated by several autocrine neuroendocrine factors including vascular endothelial growth factor-A/C (VEGF-A/C). VEGFs are secreted by several epithelia, where they modulate cell growth by autocrine and paracrine mechanisms. No data exists regarding the effect of AANAT modulation on the expressions of VEGFs by cholangiocytes. Methods In this study, we evaluated the effect of local modulation of biliary AANAT expression on the cholangiocytes synthesis of VEGF-A/C. Results The decrease in AANAT expression and subsequent lower melatonin secretion by cholangiocytes was associated with increased expression of VEGF-A/C. Overexpression of AANAT in cholangiocyte lines decreased the expression of VEGF-A/C. Conclusions Modulation of melatonin synthesis may affect the expression of VEGF-A/C by cholangiocytes and may modulate the hepatic microvascularization through the regulation of VEGF-A/C expression regulating biliary functions. PMID:24696833

  15. Antitumor effects of celecoxib in COX-2 expressing and non-expressing canine melanoma cell lines

    PubMed Central

    Seo, Kyoung-won; Coh, Ye-rin; Rebhun, Robert B.; Ahn, Jin-ok; Han, Sei-Myung; Lee, Hee-woo; Youn, Hwa-Young

    2016-01-01

    Cyclooxygenase-2 (COX-2) is a potential target for chemoprevention and cancer therapy. Celecoxib, a selective COX-2 inhibitor, inhibits cell growth of various types of human cancer including malignant melanoma. In dogs, oral malignant melanoma represents the most common oral tumor and is often a fatal disease. Therefore, there is a desperate need to develop additional therapeutic strategies. The purpose of this study was to investigate the anticancer effects of celecoxib on canine malignant melanoma cell lines that express varying levels of COX-2. Celecoxib induced a significant anti-proliferative effect in both LMeC and CMeC-1 cells. In the CMeC cells, treatment of 50 µM celecoxib caused an increase in cells in the G0/G1 and a decreased proportion of cells in G-2 phase. In the LMeC cells, 50 µM of celecoxib led to an increase in the percentage of cells in the sub-G1 phase and a significant activation of caspase-3 when compared to CMeC-1 cells. In conclusion, these results demonstrate that celecoxib exhibits antitumor effects on canine melanoma LMeC and CMeC-1 cells by induction of G1-S cell cycle arrest and apoptosis. Our data suggest that celecoxib might be effective as a chemotherapeutic agent against canine malignant melanoma. PMID:24656746

  16. MiR-224 expression increases radiation sensitivity of glioblastoma cells

    SciTech Connect

    Upraity, Shailendra; Kazi, Sadaf; Padul, Vijay; Shirsat, Neelam Vishwanath

    2014-05-30

    Highlights: • MiR-224 expression in established glioblastoma cell lines and sporadic tumor tissues is low. • Exogenous miR-224 expression was found to increase radiation sensitivity of glioblastoma cells. • MiR-224 expression brought about 55–60% reduction in API5 expression levels. • Transfection with API5 siRNA increased radiation sensitivity of glioblastoma cells. • Low miR-224 and high API5 expression correlated with worse survival of GBM patients. - Abstract: Glioblastoma (GBM) is the most common and highly aggressive primary malignant brain tumor. The intrinsic resistance of this brain tumor limits the efficacy of administered treatment like radiation therapy. In the present study, effect of miR-224 expression on growth characteristics of established GBM cell lines was analyzed. MiR-224 expression in the cell lines as well as in primary GBM tumor tissues was found to be low. Exogenous transient expression of miR-224 using either synthetic mimics or stable inducible expression using doxycycline inducible lentiviral vector carrying miR-224 gene, was found to bring about 30–55% reduction in clonogenic potential of U87 MG cells. MiR-224 expression reduced clonogenic potential of U87 MG cells by 85–90% on irradiation at a dose of 6 Gy, a dose that brought about 50% reduction in clonogenic potential in the absence of miR-224 expression. MiR-224 expression in glioblastoma cells resulted in 55–65% reduction in the expression levels of API5 gene, a known target of miR-224. Further, siRNA mediated down-regulation of API5 was also found to have radiation sensitizing effect on glioblastoma cell lines. Analysis of the Cancer Genome Atlas data showed lower miR-224 expression levels in male GBM patients to correlate with poorer survival. Higher expression levels of miR-224 target API5 also showed significant correlation with poorer survival of GBM patients. Up-regulation of miR-224 or down-regulation of its target API5 in combination with radiation therapy

  17. Increased expression of senescence markers in cystic fibrosis airways.

    PubMed

    Fischer, Bernard M; Wong, Jessica K; Degan, Simone; Kummarapurugu, Apparao B; Zheng, Shuo; Haridass, Prashamsha; Voynow, Judith A

    2013-03-15

    Cystic Fibrosis (CF) is a chronic lung disease characterized by chronic neutrophilic airway inflammation and increased levels of neutrophil elastase (NE) in the airways. We have previously reported that NE treatment triggers cell cycle arrest. Cell cycle arrest can lead to senescence, a complete loss of replicative capacity. Importantly, senescent cells can be proinflammatory and would perpetuate CF chronic inflammation. By immunohistochemistry, we evaluated whether airway sections from CF and control subjects expressed markers of senescence, including p16(INK4a) (p16), a cyclin-dependent kinase inhibitor, phospho-Histone H2A.X (γH2A.X), and phospho-checkpoint 2 kinase (phospho-Chk2), which are also DNA damage response markers. Compared with airway epithelium from control subjects, CF airway epithelium had increased levels of expression of all three senescence markers. We hypothesized that the high load of NE in the CF airway triggers epithelial senescence by upregulating expression of p16, which inhibits cyclin-dependent kinase 4 (CDK4). Normal human bronchial epithelial (NHBE) cells, cultured in air-liquid interface were treated with NE (0, 200, and 500 nM) to induce visible injury. Total cell lysates were collected and evaluated by Western analysis for p16 protein expression and CDK4 kinase activity. NE significantly increased p16 expression and decreased CDK4 kinase activity in NHBE cells. These results support the concept that NE triggers expression of senescence markers in CF airway epithelial cells. PMID:23316069

  18. Systemic alteration of cell-surface and secreted glycoprotein expression in malignant breast cancer cell lines.

    PubMed

    Timpe, Leslie C; Yen, Roger; Haste, Nicole V; Litsakos-Cheung, Christina; Yen, Ten-Yang; Macher, Bruce A

    2013-11-01

    Breast cancer cell lines express fewer transmembrane and secreted glycoproteins than nonmalignant ones. The objective of these experiments was to characterize the changes in the expression of several hundred glycoproteins quantitatively. Secreted and cell-surface glycoproteins were isolated using a glycoprotein capture protocol and then identified by tandem mass spectrometry. Glycoproteins expressed by a group of cell lines originating from malignant tumors of the breast were compared with those expressed by a nonmalignant set. The average number of spectral counts (proportional to relative protein abundance) and the total number of glycopeptides in the malignant samples were reduced to about two-thirds of the level in the nonmalignant samples. Most glycoproteins were expressed at a different level in the malignant samples, with nearly as many increasing as decreasing. The glycoproteins with reduced expression accounted for a larger change in spectral counts, and hence for the net loss of spectral counts in the malignant lines. Similar results were found when the glycoproteins were studied via identified glycosylation sites only, or through identified sites together with non-glycopeptides. The overall reduction is largely due to the loss of integrins, laminins and other proteins that form or interact with the basement membrane.

  19. Dopamine receptor activation increases glial cell line-derived neurotrophic factor in experimental stroke.

    PubMed

    Kuric, Enida; Wieloch, Tadeusz; Ruscher, Karsten

    2013-09-01

    Treatment with levodopa enhances functional recovery after experimental stroke but its mechanisms of action are elusive. Reactive astrocytes in the ischemic hemisphere are involved in mechanisms promoting recovery and also express dopamine 1 (D1) and dopamine 2 (D2) receptors. Here we investigated if the activation of astrocytic dopamine receptors (D1 and D2) regulates the expression of glial cell line-derived neurotrophic factor (GDNF) after combined in vitro hypoxia/aglycemia (H/A) and studied the expression of GDNF in the ischemic brain after treatment with levodopa/benserazide following transient occlusion of the middle cerebral artery (tMCAO) in the rat. Twenty-four hours after H/A, GDNF levels were upregulated in exposed astrocytes compared to normoxic control cultures and further elevated by the addition of the selective D1 receptor agonist (R)-(+)-SKF-38393 hydrochloride while D1 receptor antagonism by R(+)-SCH-23390 hydrochloride significantly reduced GDNF. No effect on GDNF levels was observed by the application of the D2 receptor agonist R(-)-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide hydrate or S-(-)-eticlopride hydrochloride (D2 receptor antagonist). After tMCAO, GDNF was upregulated in D1 expressing reactive astrocytes in the peri-infarct area. In addition, treatment with levodopa/benserazide significantly increased GDNF levels in the infarct core and peri-infarct area after tMCAO without affecting the expression of glial fibrillar acidic protein (GFAP), an intermediate filament and marker of reactive gliosis. After stroke, GDNF levels increase in the ischemic hemisphere in rats treated with levodopa, implicating GDNF in the mechanisms of tissue reorganization and plasticity and in l-DOPA enhanced recovery of lost brain function. Our results support levodopa treatment as a potential recovery enhancing therapy in stroke patients.

  20. Increased expression of transglutaminase 2 drives glycolytic metabolism in renal carcinoma cells.

    PubMed

    Ku, Bo Mi; Lee, Chang-Hun; Lee, Seon-Hyeong; Kim, Soo-Youl

    2014-06-01

    Transglutaminase 2 (TGase 2) expression and glycolysis are increased in most renal cell carcinoma (RCC) cell lines compared to the HEK293 kidney cell line. Although increased glycolysis and altered tricarboxylic acid cycle are common in RCC, the detailed mechanism by which this phenomenon occurs remains to be elucidated. In the present study, TGase 2 siRNA treatment lowered glucose consumption and lactate levels by about 20-30 % in RCC cells; conversely, high expression of TGase 2 increased glucose consumption and lactate production together with decreased mitochondrial aconitase (Aco 2) levels. In addition, TGase 2 siRNA increased mitochondrial membrane potential and ATP levels by about 20-30 % and restored Aco 2 levels in RCC cells. Similarly, Aco 2 levels and ATP production decreased significantly upon TGase 2 overexpression in HEK293 cells. Therefore, TGase 2 leads to depletion of Aco 2, which promotes glycolytic metabolism in RCC cells. PMID:24643363

  1. Beta-cell gene expression and functional characterisation of the human insulinoma cell line CM.

    PubMed

    Baroni, M G; Cavallo, M G; Mark, M; Monetini, L; Stoehrer, B; Pozzilli, P

    1999-04-01

    Animal insulinoma cell lines are widely used to study physiological and pathophysiological mechanisms involved in glucose metabolism and to establish in vitro models for studies on beta-cells. In contrast, human insulinoma cell lines are rarely used because of difficulties in obtaining and culturing them for long periods. The aim of our study was to investigate, under different experimental conditions, the capacity of the human insulinoma cell line CM to retain beta-cell function, particularly the expression of constitutive beta-cell genes (insulin, the glucose transporters GLUT1 and GLUT2, glucokinase), intracellular and secreted insulin, beta-cell granules, and cAMP content. Results showed that CM cells from an early-passage express specific beta-cell genes in response to glucose stimulation, in particular the insulin and GLUT genes. Such capacity is lost at later passages when cells are cultured at standard glucose concentrations. However, if cultured at lower glucose concentration (0.8 mM) for a longer time, CM cells re-acquire the capacity to respond to glucose stimulation, as shown by the increased expression of beta-cell genes (insulin, GLUT2, glucokinase). Nonetheless, insulin secretion could not be restored under such experimental conditions despite the presence of intracellular insulin, although cAMP response to a potent activator of adenylate cyclase, forskolin, was present indicating a viable system. In conclusion, these data show that the human insulinoma cell line CM, at both early-passage and late-passage, posseses a functional glucose-signalling pathway and insulin mRNA expression similar to normal beta-cells, representing, therefore, a good model for studies concerning the signalling and expression of beta-cells. Furthermore, we have previously shown that it is also a good model for immunological studies. In this respect it is important to note that the CM cell line is one of the very few existing human beta-cell lines in long-term culture.

  2. Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines

    PubMed Central

    Moinfar, Zahra; Dambach, Hannes; Schoenebeck, Bodo; Förster, Eckart; Prochnow, Nora; Faustmann, Pedro Michael

    2016-01-01

    Introduction Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43) and estrogen receptors (ERs). Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2) on Cx43 expression in two glioma cell lines with variable native expression of Cx43. Materials and Methods F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERβ and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique. Results E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERβ was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERβ in C6 cultures, while it decreased ERα expression in F98 glioma cells. Discussion These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma. PMID:26919293

  3. Bone-related matrix proteins expression in vitro and in vivo by marrow stromal cell line.

    PubMed

    Benayahu, D; Gurevitz, O A; Shamay, A

    1994-10-01

    MBA-1, a bone marrow stroma-derived cell line, was transplanted in an ectopic site and formed endochondral bone. The ossicle developed through stages of cell proliferation, differentiated into a zone of hypertrophy and formed a chondroid-like area which further developed to primary mineralized bone. We explored the expression of various matrix proteins by MBA-1 cells in vitro and in the ossicle formed in vivo. MBA-1 cells constitutively expressed mRNAs encoding for collagen I, non-collagenous proteins and alkaline phosphatase. RNA extracted from the ossicle formed by these cells was expressed in a different pattern. The in vivo maturation of MBA-1 cells was accompanied by low expression of mRNA for procollagen alpha 2(I) and a marked increase in osteonectin and osteopontin mRNA levels. Thus, the ability to follow expression of these genes through bone formation in vivo has been demonstrated. PMID:9437244

  4. Potassium increases the antitumor effects of ascorbic acid in breast cancer cell lines in vitro

    PubMed Central

    FRAJESE, GIOVANNI VANNI; BENVENUTO, MONICA; FANTINI, MASSIMO; AMBROSIN, ELENA; SACCHETTI, PAMELA; MASUELLI, LAURA; GIGANTI, MARIA GABRIELLA; MODESTI, ANDREA; BEI, ROBERTO

    2016-01-01

    Ascorbic acid (A) has been demonstrated to exhibit anti-cancer activity in association with chemotherapeutic agents. Potassium (K) is a regulator of cellular proliferation. In the present study, the biological effects of A and K bicarbonate, alone or in combination (A+K), on breast cancer cell lines were evaluated. The survival of cancer cells was determined by sulforhodamine B cell proliferation assay, while analysis of the cell cycle distribution was conducted via fluorescence-activated cell sorting. In addition, the expression of signaling proteins was analyzed upon treatment. The results indicated that there was a heterogeneous response of the different cell lines to A and K, and the best effects were achieved by A+K and A treatment. The interaction between A+K indicated an additive or synergistic effect. In addition, A+K increased the percentage of cells in the sub-G1 phase of the cell cycle, and was the most effective treatment in activating the degradation of poly(adenosine diphosphate-ribose) polymerase-1. In the breast cancer cell line MCF-7, A+K induced the appearance of the 18 kDa isoform of B-cell lymphoma-2-associated X protein (Bax), which is a more potent inducer of apoptosis than the full-length Bax-p21. The effects of A and K on the phosphorylation of extracellular signal-regulated kinase (ERK)1 and ERK2 were heterogeneous. In addition, treatment with K, A and A+K inhibited the expression of nuclear factor-κB. Overall, the results of the present study indicated that K potentiated the anti-tumoral effects of A in breast cancer cells in vitro. PMID:27313770

  5. Gene expression profiling analysis of osteosarcoma cell lines

    PubMed Central

    SUN, LU; LI, JIE; YAN, BING

    2015-01-01

    Osteosarcoma (OS) is the most common type of primary bone malignancy and has a poor prognosis. To investigate the mechanisms of osteosarcoma, the present analyzed the GSE28424 microarray. GSE28424 was downloaded from the Gene Expression Omnibus, and included a collective of 19 OS cell lines and four normal bone cell lines, which were used as controls. Subsequently, the differentially expressed genes (DEGs) were screened using the Limma package in Bioconductor. Gene Ontology (GO) and pathway enrichment analysis of the DEGs was performed using the Database for Annotation, Visualization and Integrated Discovery, interactions between the proteins encoded by the DEGs were identified using STRING, and the protein-protein interaction (PPI) network was visualized using Cytoscape. In addition, modular analysis of the PPI network was performed using the Clique Percolation Method (CPM) in CFinder. A total of 1,170 DEGs were screened, including 530 upreguated and 640 downregulated genes. The enriched functions included organelle fission, immune response and response to wounding. In addition, RPL8 was observed to be involved with the ribosomal pathway in module A of the PPI network of the DEGs. PLCG1, SYK and PLCG2 were also involved in the B-cell receptor signaling pathway in module B and the Fc-epsilon RI signaling pathway in module C. In addition, AURKA (degree=39), MAD2L1 (degree=38), CDCA8 (degree=38), BUB1 (degree=37) and MELK (degree=37) exhibited higher degrees of connectivity in module F. The results of the present study suggested that the RPL8, PLCG1, PLCG2, SYK, MAD2L1, AURKA, CDCA8, BUB1 and MELK genes may be involved in OS. PMID:26096802

  6. Modulation of homeobox gene expression alters the phenotype of human hematopoietic cell lines.

    PubMed

    Shen, W F; Detmer, K; Mathews, C H; Hack, F M; Morgan, D A; Largman, C; Lawrence, H J

    1992-03-01

    We have previously reported that certain genes of the HOX2 cluster of homeobox genes on human chromosome 17 are specifically expressed in human leukemic cell lines with erythroid potential, suggesting that these genes are involved in hematopoietic differentiation. We now show that the expression of the HOX 2.2 gene decreases during erythropoietin-induced differentiation of the erythroid cell line MB02. In order to study the role of the HOX 2.2 homeobox gene in hematopoiesis, vectors producing sense or antisense transcripts were introduced into K562 and HEL cells, pluripotent lines with erythroid and myeloid features. Overexpression of HOX 2.2 is associated with loss of erythroid features in both lines and an increase in certain myelomonocytic markers in K562 cells. Expression of antisense HOX 2.2 is associated with an increase in erythroid features in HEL cells and a mild decrease in myeloid characteristics in K562 cells. Overexpression of the adjacent HOX 2.1 gene in K562 cells does not produce similar phenotype changes. These data demonstrate that modulation of a specific HOX 2 homeobox gene can change the phenotype of somatic cells and suggest that certain HOX 2 genes play a role in blood cell differentiation.

  7. Roles of histamine on the expression of aldehyde dehydrogenase 1 in endometrioid adenocarcinoma cell line.

    PubMed

    Wang, Yi; Jiang, Yang; Ikeda, Jun-Ichiro; Tian, Tian; Sato, Atsushi; Ohtsu, Hiroshi; Morii, Eiichi

    2014-10-01

    Cancer-initiating cells (CICs) are a limited number of cells that are essential for maintenance, recurrence, and metastasis of tumors. Aldehyde dehydrogenase 1 (ALDH1) has been recognized as a marker of CICs. We previously reported that ALDH1-high cases of uterine endometrioid adenocarcinoma showed poor prognosis, and that ALDH1 high population was more tumorigenic, invasive, and resistant to apoptosis than ALDH1 low population. Histamine plays a critical role in cancer cell proliferation, migration, and invasion. Here, we examined the effect of histamine on ALDH1 expression in endometrioid adenocarcinoma cell line. The addition of histamine increased ALDH1 high population, which was consistent with the result that histamine enhanced the invasive ability and the resistance to anticancer drug. Among 4 types of histamine receptors, histamine H1 and H2 receptor (H1R and H2R) were expressed in endometrioid adenocarcinoma cell line. The addition of H1R agonist but not H2R agonist increased ALDH1. The antagonist H1R but not H2R inhibited the effect of histamine on ALDH1 expression. These results indicated that histamine increased the expression of ALDH1 via H1R but not H2R. These findings may provide the evidence for exploring a new strategy to suppress CICs by inhibiting ALDH1 expression with histamine.

  8. Prostaglandin E₂ increases fibroblast gene-specific and global DNA methylation via increased DNA methyltransferase expression.

    PubMed

    Huang, Steven K; Scruggs, Anne M; Donaghy, Jake; McEachin, Richard C; Fisher, Aaron S; Richardson, Bruce C; Peters-Golden, Marc

    2012-09-01

    Although alterations in DNA methylation patterns have been associated with specific diseases and environmental exposures, the mediators and signaling pathways that direct these changes remain understudied. The bioactive lipid mediator prostaglandin E(2) (PGE(2)) has been shown to exert a myriad of effects on cell survival, proliferation, and differentiation. Here, we report that PGE(2) also signals to increase global DNA methylation and DNA methylation machinery in fibroblasts. HumanMethylation27 BeadChip array analysis of primary fetal (IMR-90) and adult lung fibroblasts identified multiple genes that were hypermethylated in response to PGE(2). PGE(2), compared with nontreated controls, increased expression and activity (EC(50)∼10(7) M) of one specific isoform of DNA methyltransferase, DNMT3a. Silencing of DNMT3a negated the ability of PGE(2) to increase DNMT activity. The increase in DNMT3a expression was mediated by PGE(2) signaling via its E prostanoid 2 receptor and the second messenger cAMP. PGE(2), compared with the untreated control, increased the expression and activity of Sp1 and Sp3 (EC(50)∼3×10(7) M), transcription factors known to increase DNMT3a expression, and inhibition of these transcription factors abrogated the PGE(2) increase of DNMT3a expression. These findings were specific to fibroblasts, as PGE(2) decreased DNMT1 and DNMT3a expression in RAW macrophages. Taken together, these findings establish that DNA methylation is regulated by a ubiquitous bioactive endogenous mediator. Given that PGE(2) biosynthesis is modulated by environmental toxins, various disease states, and commonly used pharmacological agents, these findings uncover a novel mechanism by which alterations in DNA methylation patterns may arise in association with disease and certain environmental exposures.

  9. An Empirical Expression for the Line Widths of Ammonia

    NASA Technical Reports Server (NTRS)

    Brown, Linda R.; Peterson, Dean B.

    1994-01-01

    The hydrogen-broadened line widths of 116 (sup 14)NH(sub 3) ground state transitions have been measured at 0.006 cm(sup -1) resolution using a Bruker spectrometer in the 24 to 210 cm(sup -1) region. The rotational variation of the experimental widths with J(sup '),K(sup ') = 1,0 to 10,10 has been reproduced to 2.4 % using an heuristically derived expression of the form

    gamma = a(sub 0) + a(sub 1) J(sup ') + a (sub 2) K(sup ') + a(sub 3) J(sup ')(sup 2) + a(sub 4) J(sup ') K(sup ')

    where J(sup ') and K(sup ') are the lower state symmetric top quantum numbers. This function has also been applied to the measured widths of the 58 transitions of nu(sub 1) at 3 (micro)m, each broadened by N(sub 2), O(sub 2), Ar, H(sub 2), and He. The rms of the observed minus calculated widths are 5% or better for the five foreign broadeners. The values of the fitted constants suggest that for some broadeners the expression might also be written as

    gamma = a(sub 0) + b(sub 1) J(sup ') + b(sub 2)(J(sup ' )- K(sup ')) + b(sub 3) J(sup ')(J(sup ') - K(sup '))

    .

  10. Increased intra- and extracellular granzyme expression in patients with tuberculosis.

    PubMed

    Garcia-Laorden, M Isabel; Blok, Dana C; Kager, Liesbeth M; Hoogendijk, Arie J; van Mierlo, Gerard J; Lede, Ivar O; Rahman, Wahid; Afroz, Rumana; Ghose, Aniruddha; Visser, Caroline E; Md Zahed, Abu Shahed; Husain, Md Anwar; Alam, Khan Mashrequl; Chandra Barua, Pravat; Hassan, Mahtabuddin; Hossain, Ahmed; Tayab, Md Abu; Day, Nick; Dondorp, Arjen M; de Vos, Alex F; van der Poll, Tom

    2015-09-01

    Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Granzymes (gzms) are proteases mainly found in cytotoxic lymphocytes, but also extracellularly. While the role of gzms in target cell death has been widely characterized, considerable evidence points towards broader roles related to infectious and inflammatory responses. To investigate the expression of the gzms in TB, intracellular gzms A, B and K were measured by flow cytometry in lymphocyte populations from peripheral blood mononuclear cells from 18 TB patients and 12 healthy donors from Bangladesh, and extracellular levels of gzmA and B were measured in serum from 58 TB patients and 31 healthy controls. TB patients showed increased expression of gzmA in CD8(+) T, CD4(+) T and CD56(+) T, but not NK, cells, and of gzmB in CD8(+) T cells, when compared to controls. GzmK expression was not altered in TB patients in any lymphocyte subset. The extracellular levels of gzmA and, to a lesser extent, of gzmB, were increased in TB patients, but did not correlate with intracellular gzm expression in lymphocyte subsets. Our results reveal enhanced intra- and extracellular expression of gzmA and B in patients with pulmonary TB, suggesting that gzms are part of the host response to tuberculosis.

  11. Increased expression and aberrant localization of mucin 13 in metastatic colon cancer.

    PubMed

    Gupta, Brij K; Maher, Diane M; Ebeling, Mara C; Sundram, Vasudha; Koch, Michael D; Lynch, Douglas W; Bohlmeyer, Teresa; Watanabe, Akira; Aburatani, Hiroyuki; Puumala, Susan E; Jaggi, Meena; Chauhan, Subhash C

    2012-11-01

    MUC13 is a newly identified transmembrane mucin. Although MUC13 is known to be overexpressed in ovarian and gastric cancers, limited information is available regarding the expression of MUC13 in metastatic colon cancer. Herein, we investigated the expression profile of MUC13 in colon cancer using a novel anti-MUC13 monoclonal antibody (MAb, clone ppz0020) by immunohistochemical (IHC) analysis. A cohort of colon cancer samples and tissue microarrays containing adjacent normal, non-metastatic colon cancer, metastatic colon cancer, and liver metastasis tissues was used in this study to investigate the expression pattern of MUC13. IHC analysis revealed significantly higher (p<0.001) MUC13 expression in non-metastatic colon cancer samples compared with faint or very low expression in adjacent normal tissues. Interestingly, metastatic colon cancer and liver metastasis tissue samples demonstrated significantly (p<0.05) higher cytoplasmic and nuclear MUC13 expression compared with non-metastatic colon cancer and adjacent normal colon samples. Moreover, cytoplasmic and nuclear MUC13 expression correlated with larger and poorly differentiated tumors. Four of six tested colon cancer cell lines also expressed MUC13 at RNA and protein levels. These studies demonstrate a significant increase in MUC13 expression in metastatic colon cancer and suggest a correlation between aberrant MUC13 localization (cytoplasmic and nuclear expression) and metastatic colon cancer.

  12. SFRP2 Is Associated with Increased Adiposity and VEGF Expression

    PubMed Central

    Crowley, Rachel K.; Bujalska, Iwona J.; Hassan-Smith, Zaki K.; Hazlehurst, Jonathan M.; Foucault, Danielle R.; Stewart, Paul M.; Tomlinson, Jeremy W.

    2016-01-01

    Aims The aim of this study was to assess depot-specific expression and secretion of secreted frizzled-related protein 2 (sFRP2) by adipose tissue and its effect on adipocyte biology. We measured serum sFRP2 concentrations in 106 patients in vivo to explore its relationship to fat mass, glycaemia and insulin resistance. Methods Expression of sFRP2 in mouse and human tissues was assessed using polymerase chain reaction and Western blot. Western blot confirmed secretion of sFRP2 by adipose tissue into cell culture medium. Effects of recombinant sFRP2 on lipogenesis and preadipocyte proliferation were measured. Preadipocyte expression of the angiogenic genes vascular endothelial growth factor (VEGF) and nuclear factor of activated T-cells 3 (NFATC3) was measured after recombinant sFRP2 exposure. Complementary clinical studies correlating human serum sFRP2 with age, gender, adiposity and insulin secretion were also performed. Results sFRP2 messenger RNA (mRNA) was expressed in mouse and human adipose tissue. In humans, sFRP2 mRNA expression was 4.2-fold higher in omental than subcutaneous adipose. Omental adipose tissue secreted 63% more sFRP2 protein than subcutaneous. Treatment with recombinant sFRP2 did not impact on lipogenesis or preadipocyte proliferation but was associated with increased VEGF mRNA expression. In human subjects, circulating insulin levels positively correlated with serum sFRP2, and levels were higher in patients with abnormal glucose tolerance (34.2ng/ml) compared to controls (29.5ng/ml). A positive correlation between sFRP2 and BMI was also observed. Conclusions Circulating sFRP2 is associated with adipose tissue mass and has a potential role to drive adipose angiogenesis through enhanced VEGF expression. PMID:27685706

  13. Ethynylestradiol increases expression and activity of rat liver MRP3.

    PubMed

    Ruiz, María L; Villanueva, Silvina S M; Luquita, Marcelo G; Vore, Mary; Mottino, Aldo D; Catania, Viviana A

    2006-06-01

    We evaluated the effect of ethynylestradiol (EE) administration (5 mg/kg b.wt. s.c., for 5 consecutive days) on the expression and activity of multidrug resistance-associated protein 3 (Mrp3) in rats. Western blotting analysis revealed decreased Mrp2 (-41%) and increased Mrp3 (+200%) expression by EE. To determine the functional impact of up-regulation of Mrp3 versus Mrp2, we measured the excretion of acetaminophen glucuronide (APAP-glu), a common substrate for both transporters, into bile and perfusate in the recirculating isolated perfused liver (IPL) model. APAP-glu was generated endogenously from acetaminophen (APAP), which was administered as a tracer dose (2 micromol/ml) into the perfusate. Biliary excretion of APAP-glu after 60 min of perfusion was reduced in EE-treated rats (-80%). In contrast, excretion into the perfusate was increased by EE (+45%). Liver content of APAP-glu at the end of the experiment was reduced by 36% in the EE group. The total amount of glucuronide remained the same in both groups. Taken together, these results indicate that up-regulation of Mrp3 led to an exacerbated basolateral versus canalicular excretion of conjugated APAP in IPL. We conclude that induced expression of basolateral Mrp3 by EE may represent a compensatory mechanism to prevent intracellular accumulation of common Mrp substrates, either endogenous or exogenous, due to reduced expression and activity of apical Mrp2. PMID:16554369

  14. Exercise-Mediated Increase in Nigral Tyrosine Hydroxylase Is Accompanied by Increased Nigral GFR-α1 and EAAC1 Expression in Aging Rats.

    PubMed

    Arnold, Jennifer C; Salvatore, Michael F

    2016-02-17

    Exercise may alleviate locomotor impairment in Parkinson's disease (PD) or aging. Identifying molecular responses immediately engaged by exercise in the nigrostriatal pathway and allied tissue may reveal critical targets associated with its long-term benefits. In aging, there is loss of tyrosine hydroxylase (TH) and the glial cell line-derived neurotrophic factor (GDNF) receptor, GFR-α1, in the substantia nigra (SN). Exercise can increase GDNF expression, but its effect on GFR-α1 expression is unknown. Infusion of GDNF into striatum or GFR-α1 in SN, respectively, can increase locomotor activity and TH function in SN but not striatum in aged rats. GDNF may also increase glutamate transporter expression, which attenuates TH loss in PD models. We utilized a footshock-free treadmill exercise regimen to determine the immediate impact of short-term exercise on GFR-α1 expression, dopamine regulation, glutamate transporter expression, and glutamate uptake in 18 month old male Brown-Norway/Fischer 344 F1 hybrid rats. GFR-α1 and TH expression significantly increased in SN but not striatum. This exercise regimen did not affect glutamate uptake or glutamate transporter expression in striatum. However, EAAC1 expression increased in SN. These results indicate that nigral GFR-α1 and EAAC1 expression increased in conjunction with increased nigral TH expression following short-term exercise.

  15. Sulindac sulfide selectively increases sensitivity of ABCC1 expressing tumor cells to doxorubicin and glutathione depletion

    PubMed Central

    Whitt, Jason D.; Keeton, Adam B.; Gary, Bernard D.; Sklar, Larry A.; Sodani, Kamlesh; Chen, Zhe-Sheng; Piazza, Gary A.

    2016-01-01

    Abstract ATP-binding cassette (ABC) transpo rters ABCC1 (MRP1), ABCB1 (P-gp), and ABCG2 (BCRP) contribute to chemotherapy failure. The primary goals of this study were to characterize the efficacy and mechanism of the non­steroidal anti-inflammatory drug (NSAID), sulindac sulfide, to reverse ABCC1 mediated resistance to chemother­apeutic drugs and to determine if sulindac sulfide can influence sensitivity to chemotherapeutic drugs independently of drug efflux. Cytotoxicity assays were performed to measure resistance of ABC-expressing cell lines to doxoru­bicin and other chemotherapeutic drugs. NSAIDs were tested for the ability to restore sensitivity to resistance selected tumor cell lines, as well as a large panel of standard tumor cell lines. Other experiments characterized the mechanism by which sulindac sulfide inhibits ABCC1 substrate and co-substrate (GSH) transport in isolated membrane vesicles and intact cells. Selective reversal of multi-drug resistance (MDR), decreased efflux of doxor­ubicin, and fluorescent substrates were demonstrated by sulindac sulfide and a related NSAID, indomethacin, in resistance selected and engineered cell lines expressing ABCC1, but not ABCB1 or ABCG2. Sulindac sulfide also inhibited transport of leukotriene C4 into membrane vesicles. Sulindac sulfide enhanced the sensitivity to doxoru­bicin in 24 of 47 tumor cell lines, including all melanoma lines tested (7-7). Sulindac sulfide also decreased intra­cellular GSH in ABCC1 expressing cells, while the glutathione synthesis inhibitor, BSO, selectively increased sensitivity to sulindac sulfide induced cytotoxicity. Sulindac sulfide potently and selectively reverses ABCC1-mediated MDR at clinically achievable concentrations. ABCC1 expressing tumors may be highly sensitive to the direct cytotoxicity of sulindac sulfide, and in combination with chemotherapeutic drugs that induce oxidative stress.

  16. Increased Expression of the dsRNA-Activated Protein Kinase PKR in Breast Cancer Promotes Sensitivity to Doxorubicin

    PubMed Central

    Bennett, Richard L.; Carruthers, Aubrey L.; Hui, Teng; Kerney, Krystal R.; Liu, Xiangfei; May, W. Stratford

    2012-01-01

    It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H2O2-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression

  17. Relative quantification of beta-casein expression in primary goat mammary epithelial cell lines.

    PubMed

    Ogorevc, J; Dovč, P

    2015-04-15

    Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

  18. Chemoresistance of CD133(+) colon cancer may be related with increased survivin expression.

    PubMed

    Lee, Mi-Ra; Ji, Sun-Young; Mia-Jan, Khalilullah; Cho, Mee-Yon

    2015-07-31

    CD133, putative cancer stem cell marker, deemed to aid chemoresistance. However, this claim has been challenged recently and we previously reported that patients with CD133(+) colon cancer have benefit from 5-fluorouracil (5-FU) chemotherapy incontrast to no benefit in patients with CD133(-) cancer. To elucidate the role of CD133 expression in chemoresistance, we silenced the CD133 expression in a colon cancer cell line and determined its effect on the biological characteristics downstream. We comparatively analyzed the sequential changes of MDR1, ABCG2, AKT1 and survivin expression and the result of proliferation assay (WST-1 assay) with 5-FU treatment in CD133(+) and siRNA-induced CD133(-) cells, derived from Caco-2 colon cancer cell line. 5-FU treatment induced significantly increase of the mRNA expression of MDR1, ABCG2 and AKT1genes, but not protein level. CD133 had little to no effect on the mRNA and protein expression of these genes. However, survivin expression at mRNA and protein level were significantly increased in CD133(+) cells compared with siRNA-induced CD133-cells and Mock (not sorted CD133(+) cells) at 96 h after siRNA transfection. The cytotoxicity assay demonstrated notable increase of chemoresistance to 5-FU treatment (10 μM) in CD133(+) cells at 96 h after siRNA transfection. From this study, we conclude that CD133(+) cells may have chemoresistance to 5-FU through the mechanism which is related with survivin expression, instead of MDR1, ABCG2 and AKT1 expression. Therefore a survivin inhibitor can be a new target for effective treatment of CD133(+) colon cancer.

  19. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    PubMed Central

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S.; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G.; Beazely, Michael A.

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands. PMID:25426041

  20. Tetracyclines increase lipid phosphate phosphatase expression on plasma membranes and turnover of plasma lysophosphatidate.

    PubMed

    Tang, Xiaoyun; Zhao, Yuan Y; Dewald, Jay; Curtis, Jonathan M; Brindley, David N

    2016-04-01

    Extracellular lysophosphatidate and sphingosine 1-phosphate (S1P) are important bioactive lipids, which signal through G-protein-coupled receptors to stimulate cell growth and survival. The lysophosphatidate and S1P signals are terminated partly by degradation through three broad-specificity lipid phosphate phosphatases (LPPs) on the cell surface. Significantly, the expression of LPP1 and LPP3 is decreased in many cancers, and this increases the impact of lysophosphatidate and S1P signaling. However, relatively little is known about the physiological or pharmacological regulation of the expression of the different LPPs. We now show that treating several malignant and nonmalignant cell lines with 1 μg/ml tetracycline, doxycycline, or minocycline significantly increased the extracellular degradation of lysophosphatidate. S1P degradation was also increased in cells that expressed high LPP3 activity. These results depended on an increase in the stabilities of the three LPPs and increased expression on the plasma membrane. We tested the physiological significance of these results and showed that treating rats with doxycycline accelerated the clearance of lysophosphatidate, but not S1P, from the circulation. However, administering 100 mg/kg/day doxycycline to mice decreased plasma concentrations of lysophosphatidate and S1P. This study demonstrates a completely new property of tetracyclines in increasing the plasma membrane expression of the LPPs.

  1. IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells.

    PubMed

    Long, Jun; Zhang, Xulong; Wen, Mingjie; Kong, Qingli; Lv, Zhe; An, Yunqing; Wei, Xiao-Qing

    2013-01-01

    Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.

  2. Regulation of ionic conductances and gene expression by hypoxia in an oxygen sensitive cell line.

    PubMed

    Millhorn, D E; Conforti, L; Beitner-Johnson, D; Zhu, W; Raymond, R; Filisko, T; Kobayashi, S; Peng, M; Genter, M B

    1996-01-01

    We have shown that the PC12 cell line is an excellent model system for investigations of the molecular and cellular processes involved in O2-chemosensitivity. We have identified an O2-sensitive K channel in this cell line that mediates membrane depolarization, an increase in intracellular free Ca2+, and dopamine release during hypoxia. We also presented evidence which shows that expression of the gene for tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis, is stimulated by reduced O2 tension in PC12 and type I carotid body cells. In addition, we have successfully identified the DNA sequences and trans-acting protein factors that regulate transcription of the TH gene during hypoxia. The mechanisms by which a reduction in O2 tension is transduced into alter cell function including increased gene expression remain unknown. Unpublished results from our laboratory show that the increased TH gene expression during hypoxia does not require activation of the cAMP-PKA signal transduction pathway. We propose that the increase in intracellular free Ca2+ that occurs as a result of membrane depolarization might play an important role. Preliminary findings from our laboratory show that blockade of the voltage operated Ca2+ channel or chelation of intracellular Ca2+ prevent full activation of the TH gene during hypoxia. PMID:9030290

  3. IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines.

    PubMed

    Ho, Steven C L; Bardor, Muriel; Feng, Huatao; Mariati; Tong, Yen Wah; Song, Zhiwei; Yap, Miranda G S; Yang, Yuansheng

    2012-01-01

    A Tricistronic vector utilizing internal ribosome entry site (IRES) elements to express the light chain (LC), heavy chain (HC), and a neomycin phosphotransferase (NPT) selection marker from one transcript is designed for generation of mAb expressing CHO cell lines. As compared to the commonly used vectors, benefits of this design include: (1) minimized non-expressing clones, (2) enhanced stable mAb productivity without gene amplification, (3) control of LC and HC expression at defined ratios, and (4) consistent product quality. After optimization of the LC and HC arrangement and increasing selection stringency by weakening the NPT selection marker, this Tricistronic vector is able to generate stably transfected pools with specific productivity (qmAb) greater than 5pg/cell/day (pcd) and titers over 150mg/L. 5% of clones from these pools have qmAb greater than 20pcd and titers ranging from 300 to more than 500mg/L under non-optimized shake flask batch cultures using commercially available protein-free medium. The mAb produced by these clones have low aggregation and consistent glycosylation profiles. The entire process of transfection to high-expressing clones requires only 6 months. The IRES-mediated Tricistronic vector provides an attractive alternative to commonly used vectors for fast generation of mAb CHO cell lines with high productivity. PMID:22024589

  4. Matrix metalloproteinase gene expressions might be oxidative stress targets in gastric cancer cell lines

    PubMed Central

    Gencer, Salih; Cebeci, Anil

    2013-01-01

    Objective Oxidative stress is linked to increased risk of gastric cancer and matrix metalloproteinases (MMPs) are important in the invasion and metastasis of gastric cancer. We aimed to analyze the effect of the accumulation of oxidative stress in the gastric cancer MKN-45 and 23132/87 cells following hydrogen peroxide (H2O2) exposure on the expression patterns of MMP-1, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-12, MMP-14, MMP-15, MMP-17, MMP-23, MMP-28, and β-catenin genes. Methods The mRNA transcripts in the cells were determined by RT-PCR. Following H2O2 exposure, oxidative stress in the viable cells was analyzed by 2',7'-dichlorofluorescein diacetate (DCFH-DA). Caffeic acid phenethyl ester (CAPE) was used to eliminate oxidative stress and the consequence of H2O2 exposure and its removal on the expressions of the genes were evaluated by quantitative real-time PCR. Results The expressions of MMP-1, MMP-7, MMP-14, MMP-15, MMP-17 and β-catenin in MKN-45 cells and only the expression of MMP-15 in 23132/87 cells were increased. Removal of the oxidative stress resulted in decrease in the expressions of MMP genes of which the expressions were increased after H2O2 exposure. β-catenin, a transcription factor for many genes including MMPs, also displayed decreased levels of expression in both of the cell lines following CAPE treatment. Conclusions Our data suggest that there is a remarkable link between the accumulation of oxidative stress and the increased expressions of MMP genes in the gastric cancer cells and MMPs should be considered as potential targets of therapy in gastric cancers due to its continuous exposure to oxidative stress. PMID:23825909

  5. Increased expression of the antiapoptotic protein MCL1 in canine mast cell tumors.

    PubMed

    Amagai, Yosuke; Tanaka, Akane; Matsuda, Akira; Oida, Kumiko; Jung, Kyungsook; Nishikawa, Sho; Jang, Hyosun; Ishizaka, Saori; Matsuda, Hiroshi

    2013-07-31

    Myeloid cell leukemia sequence 1 (MCL1) is a potent antiapoptotic protein that plays a critical role in cell survival and drug resistance in various cancers. However, to the best of our knowledge, the role of MCL1 in mast cell tumors (MCTs) has not been investigated in dogs. Here, we detected increased MCL1 expression in MCT cell lines, regardless of the presence of a c-kit mutation. MCL1 expression increased when the cells were exposed to specific inhibitors of mitogen-activated protein kinase or Janus kinase-signaling pathways, thus protecting the cells from apoptosis, but not when KIT or phosphatidylinositol-3 kinase signaling cascades were inhibited. These results indicate that MCL1 expression may contribute to MCT survival and confer drug resistance. PMID:23428776

  6. Concurrent expression of heme oxygenase-1 and p53 in human retinal pigment epithelial cell line

    SciTech Connect

    Lee, Sang Yull; Jo, Hong Jae; Kim, Kang Mi; Song, Ju Dong; Chung, Hun Taeg; Park, Young Chul

    2008-01-25

    Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. Here, we investigated the effects of HO activity on the expression of p53 in the human retinal pigment epithelium (RPE) cell line ARPE-19. Cobalt protoporphyrin (CoPP) induced the expression of both HO-1 and p53 without significant toxicity to the cells. In addition, the blockage of HO activity with the iron chelator DFO or with HO-1 siRNA inhibited the CoPP-induced expression of p53. Similarly, zinc protoporphyrin (ZnPP), an inhibitor of HO, suppressed p53 expression in ARPE-19 cells, although ZnPP increased the level of HO-1 protein while inhibiting HO activity. Also, CoPP-induced p53 expression was not affected by the formation of reactive oxygen species (ROS). Based on these results, we conclude that HO activity is involved in the regulation of p53 expression in a ROS-independent mechanism, and also suggest that the expression of p53 in ARPE-19 cells is associated with heme metabolites such as biliverdin/bilirubin, carbon monoxide, and iron produced by the activity of HO.

  7. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  8. Interleukin-induced increase in Ia expression by normal mouse B cells

    PubMed Central

    1984-01-01

    The constitutive culture supernatant (SN) of the macrophage tumor line P388D1 (P388 SN) and the concanavalin A (Con A)-induced culture supernatant of the T cell hybridoma FS6-14.13 (FS6 Con A SN) were shown to contain nonspecific factors capable of inducing increased Ia expression by normal resting B cells in a dose-dependent manner. In six consecutive experiments the relative increase in Ia expression induced by P388 SN was 4.9 +/- 0.9, with FS6 Con A SN 10.7 +/- 1.5, and with a combination of both preparations 13.0 +/- 1.7. This increase in Ia expression was observed to occur in virtually all the B cells, reaching maximum levels within 24 h of culture. The interleukin-induced increase in B cell Ia expression occurred in the absence of ancillary signals provided by ligand-receptor Ig cross-linking and despite the fact that virtually all the control B cells, cultured in the absence of factors, remained in G0. These results suggest that functional receptors for at least some interleukins are expressed on normal resting B cells and their effects can be manifest in the absence of additional activating signals. The increased Ia expression induced by the nonspecific factor preparations was shown to be correlated with enhanced antigen- presenting capacity by the B cells to T cell hybridomas. The nature of the interleukins responsible for these effects remains to be definitively determined, however, the activity of FS6 Con A SN was shown to correlate with B cell growth factor activity and increased B cell Ia expression was not observed using interleukin 2 (IL-2) or interferon-gamma, prepared by recombinant DNA technology. PMID:6432933

  9. Chemoresistance of CD133{sup +} colon cancer may be related with increased survivin expression

    SciTech Connect

    Lee, Mi-Ra; Ji, Sun-Young; Mia-Jan, Khalilullah; Cho, Mee-Yon

    2015-07-31

    CD133, putative cancer stem cell marker, deemed to aid chemoresistance. However, this claim has been challenged recently and we previously reported that patients with CD133{sup +} colon cancer have benefit from 5-fluorouracil (5-FU) chemotherapy incontrast to no benefit in patients with CD133{sup −} cancer. To elucidate the role of CD133 expression in chemoresistance, we silenced the CD133 expression in a colon cancer cell line and determined its effect on the biological characteristics downstream. We comparatively analyzed the sequential changes of MDR1, ABCG2, AKT1 and survivin expression and the result of proliferation assay (WST-1 assay) with 5-FU treatment in CD133{sup +} and siRNA-induced CD133{sup −} cells, derived from Caco-2 colon cancer cell line. 5-FU treatment induced significantly increase of the mRNA expression of MDR1, ABCG2 and AKT1genes, but not protein level. CD133 had little to no effect on the mRNA and protein expression of these genes. However, survivin expression at mRNA and protein level were significantly increased in CD133{sup +} cells compared with siRNA-induced CD133-cells and Mock (not sorted CD133{sup +} cells) at 96 h after siRNA transfection. The cytotoxicity assay demonstrated notable increase of chemoresistance to 5-FU treatment (10 μM) in CD133{sup +} cells at 96 h after siRNA transfection. From this study, we conclude that CD133{sup +} cells may have chemoresistance to 5-FU through the mechanism which is related with survivin expression, instead of MDR1, ABCG2 and AKT1 expression. Therefore a survivin inhibitor can be a new target for effective treatment of CD133{sup +} colon cancer. - Highlights: • We evaluate the role of CD133 in chemoresistance of colon cancer. • We compared the chemoresistance of CD133{sup +} cells and siRNA-induced CD133{sup −} cells. • CD133 had little to no effect on MDR1, ABCG2 and AKT1 expression. • Survivin expression and chemoresistance were increased in CD133{sup +} colon cancer cells.

  10. Conceptus development during blastocyst elongation in lines of pigs selected for increased uterine capacity or ovulation rate.

    PubMed

    Miles, J R; Freking, B A; Blomberg, L A; Vallet, J L; Zuelke, K A

    2008-09-01

    Lines of pigs selected for increased uterine capacity have improved conceptus survival, whereas pigs selected for increased ovulation rate have decreased conceptus survival relative to an unselected control line. The objective of this study was to evaluate conceptus development during blastocyst elongation as a potential contributing factor to differences in conceptus survival rate among these pig lines. Conceptuses were recovered from pregnant control, uterine capacity, and ovulation rate line gilts at d 10 and 12 of gestation. At d 10 of gestation, conceptus morphologic diversity was assessed by comparing within-litter average conceptus diameter and the standard deviation of conceptus diameters. At d 12 of gestation, conceptus morphologic diversity was assessed by comparing blastocyst populations obtained from individual gilts. Real-time PCR analyses for transcripts involved in steroidogenesis, cellular differentiation, and immune responsiveness were performed on spherical, ovoid, and filamentous conceptuses recovered from these selection lines. Uterine flushings were also assayed for total protein and estradiol-17beta at d 10 and 12 of gestation. Morphological data were analyzed using ANOVA with the fixed effects of line, farrowing season, and their interactions. Conceptus mortality, uterine flushing, and real-time PCR data were analyzed using ANOVA with the fixed effects of line, day or blastocyst morphology, farrowing season, and their interactions. Conceptus mortality, measured as the ratio of conceptus recovery to ovulation rate, was not different between the lines on d 10 and 12 of gestation. There were no significant line effects for conceptus morphologic diversity at d 10 and 12 of gestation. Expression of transcripts associated with steroidogenesis (steroidogenic acute regulatory protein, cytochrome P450 side chain cleavage, and aromatase), cellular differentiation (cytokeratin-18 and vimentin), and immune responsiveness (interleukin-1beta) in spherical

  11. Human MiR-544a Modulates SELK Expression in Hepatocarcinoma Cell Lines.

    PubMed

    Potenza, Nicoletta; Castiello, Filomena; Panella, Marta; Colonna, Giovanni; Ciliberto, Gennaro; Russo, Aniello; Costantini, Susan

    2016-01-01

    Hepatocellular carcinoma (HCC) is a multi-factorial cancer with a very poor prognosis; therefore, there are several investigations aimed at the comprehension of the molecular mechanisms leading to development and progression of HCC and at the definition of new therapeutic strategies. We have recently evaluated the expression of selenoproteins in HCC cell lines in comparison with normal hepatocytes. Recent results have shown that some of them are down- and others up-regulated, including the selenoprotein K (SELK), whose expression was also induced by sodium selenite treatment on cells. However, so far very few studies have been dedicated to a possible effect of microRNAs on the expression of selenoproteins and their implication in HCC. In this study, the analysis of SELK 3'UTR by bioinformatics tools led to the identification of eight sites potentially targeted by human microRNAs. They were then subjected to a validation test based on luciferase reporter constructs transfected in HCC cell lines. In this functional screening, miR-544a was able to interact with SELK 3'UTR suppressing the reporter activity. Transfection of a miR-544a mimic or inhibitor was then shown to decrease or increase, respectively, the translation of the endogenous SELK mRNA. Intriguingly, miR-544a expression was found to be modulated by selenium treatment, suggesting a possible role in SELK induction by selenium. PMID:27275761

  12. Human MiR-544a Modulates SELK Expression in Hepatocarcinoma Cell Lines

    PubMed Central

    Potenza, Nicoletta; Castiello, Filomena; Panella, Marta; Colonna, Giovanni; Ciliberto, Gennaro; Russo, Aniello; Costantini, Susan

    2016-01-01

    Hepatocellular carcinoma (HCC) is a multi-factorial cancer with a very poor prognosis; therefore, there are several investigations aimed at the comprehension of the molecular mechanisms leading to development and progression of HCC and at the definition of new therapeutic strategies. We have recently evaluated the expression of selenoproteins in HCC cell lines in comparison with normal hepatocytes. Recent results have shown that some of them are down- and others up-regulated, including the selenoprotein K (SELK), whose expression was also induced by sodium selenite treatment on cells. However, so far very few studies have been dedicated to a possible effect of microRNAs on the expression of selenoproteins and their implication in HCC. In this study, the analysis of SELK 3’UTR by bioinformatics tools led to the identification of eight sites potentially targeted by human microRNAs. They were then subjected to a validation test based on luciferase reporter constructs transfected in HCC cell lines. In this functional screening, miR-544a was able to interact with SELK 3’UTR suppressing the reporter activity. Transfection of a miR-544a mimic or inhibitor was then shown to decrease or increase, respectively, the translation of the endogenous SELK mRNA. Intriguingly, miR-544a expression was found to be modulated by selenium treatment, suggesting a possible role in SELK induction by selenium. PMID:27275761

  13. Extracellular matrix proteins expression profiling in chemoresistant variants of the A2780 ovarian cancer cell line.

    PubMed

    Januchowski, Radosław; Zawierucha, Piotr; Ruciński, Marcin; Nowicki, Michał; Zabel, Maciej

    2014-01-01

    Ovarian cancer is the leading cause of death among gynaecological malignancies. Extracellular matrix (ECM) can affect drug resistance by preventing the penetration of the drug into cancer cells and increased resistance to apoptosis. This study demonstrates alterations in the expression levels of ECM components and related genes in cisplatin-, doxorubicin-, topotecan-, and paclitaxel-resistant variants of the A2780 ovarian cancer cell line. Affymetrix Gene Chip Human Genome Array Strips were used for hybridisations. The genes that had altered expression levels in drug-resistant sublines were selected and filtered by scatter plots. The genes that were up- or downregulated more than fivefold were selected and listed. Among the investigated genes, 28 genes were upregulated, 10 genes were downregulated, and two genes were down- or upregulated depending on the cell line. Between upregulated genes 12 were upregulated very significantly--over 20-fold. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. The expression profiles of investigated genes provide a preliminary insight into the relationship between drug resistance and the expression of ECM components. Identifying correlations between investigated genes and drug resistance will require further analysis.

  14. Increased Expression of Interleukin-18 in Lenses of Ovariectomized Rats.

    PubMed

    Nagai, Noriaki; Ogata, Fumihiko; Kawasaki, Naohito; Ito, Yoshimasa

    2016-01-01

    Previous studies showed an increased prevalence of cataracts in postmenopausal women. In this study, we investigated changes in the levels of calcium ion (Ca(2+)) and interleukin (IL)-18, which are factors in cataract development, in the lenses of ovariectomized (OVX) rats, a model of postmenopausal woman. Although the Ca(2+) content in the blood of OVX rats increased 1 month after ovariectomy and subsequently decreased, the Ca(2+) content in the lenses was unchanged in OVX rats 1-3 months after ovariectomy. The Ca(2+)-ATPase activity in the lenses of OVX rats peaked 1 month after ovariectomy, and the behavior of Ca(2+)-ATPase activity in lenses of OVX rats was similar to that of the Ca(2+) concentration in the blood. It is possible that hypercalcemia increases the Ca(2+) inflow into the lens; however, the enhanced Ca(2+)-ATPase activity prevents the Ca(2+) level from rising. On the other hand, we found that the levels of both IL-18 and interferon (IFN)-γ in the lenses of OVX rats were significantly increased as compared with the lenses of sham (control) rats during the period 1-3 months after surgery. These results suggest that the expression of IFN-γ via IL-18 in the lenses of OVX rats is induced by ovariectomy, and that excessive IL-18 and IFN-γ production in the lenses may be related to cataract development in postmenopausal women. These findings support those of previous studies that assessed lens opacification in postmenopausal women.

  15. Expression of HOX genes in acute leukemia cell lines with and without MLL translocations.

    PubMed

    Quentmeier, Hilmar; Dirks, Wilhelm G; Macleod, Roderick A F; Reinhardt, Julia; Zaborski, Margarete; Drexler, Hans G

    2004-03-01

    In primary cells from acute leukemia patients, expression of the genes MEIS1, HOXA5, HOXA7 and HOXA9 has been reported to be correlated with the occurrence of MLL translocations. It was our aim to find out whether MLL mutant (MLLmu) and MLL wild-type (MLLwt) acute leukemia-derived cell lines might likewise be discriminated on the basis of HOX gene expression. Southern blot analysis, performed to verify the MLL status of the cells, showed that NOMO-1 was the only cell line not tested previously carrying a rearranged MLL gene. Fluorescence in situ hybridization analysis demonstrated that this cell line exhibited a reciprocal t(9;11)(q23;p22). Sequencing of RT-PCR products thereof identified unique MLL exon 10/AF-9 exon 5 fusion transcripts. We divided the acute leukemia-derived cell lines (n = 37) according to the results of Southern blot analysis into MLLmu (n = 19) and MLLwt (n = 18). Expression of HOX genes was then analyzed by applying reverse transcriptase-polymerase chain reaction, Northern and Western blot analyses. Acute myeloid leukemia (AML) cell lines expressed the HOX genes significantly more often than acute lymphoblastic (ALL) cell lines. In ALL, cells with MLL translocations expressed the genes 4 times more often than MLLwt cells. Most distinct was the correlation between MLL status and MEIS1 expression in ALL-derived cell lines: 8/8 MLLmu but 0/10 MLLwt cell lines expressed MEIS1. Northern and Western blot analysis confirmed that also HOXA9 and FLT3 were significantly more often and stronger expressed in MLLmu than in MLLwt ALL cell lines. These results suggest that MLL aberrations may regulate MEIS1 and HOXA9 gene expression in ALL-derived cell lines, while AML-derived cell lines express these genes independently of the MLL status. PMID:15160920

  16. Characterization of MTAP Gene Expression in Breast Cancer Patients and Cell Lines

    PubMed Central

    de Oliveira, Sarah Franco Vieira; Ganzinelli, Monica; Chilà, Rosaria; Serino, Leandro; Maciel, Marcos Euzébio; Urban, Cícero de Andrade; de Lima, Rubens Silveira; Cavalli, Iglenir João; Generali, Daniele; Broggini, Massimo

    2016-01-01

    MTAP is a ubiquitously expressed gene important for adenine and methionine salvage. The gene is located at 9p21, a chromosome region often deleted in breast carcinomas, similar to CDKN2A, a recognized tumor suppressor gene. Several research groups have shown that MTAP acts as a tumor suppressor, and some therapeutic approaches were proposed based on a tumors´ MTAP status. We analyzed MTAP and CDKN2A gene (RT-qPCR) and protein (western-blotting) expression in seven breast cancer cell lines and evaluated their promoter methylation patterns to better characterize the contribution of these genes to breast cancer. Cytotoxicity assays with inhibitors of de novo adenine synthesis (5-FU, AZA and MTX) after MTAP gene knockdown showed an increased sensitivity, mainly to 5-FU. MTAP expression was also evaluated in two groups of samples from breast cancer patients, fresh tumors and paired normal breast tissue, and from formalin-fixed paraffin embedded (FFPE) core breast cancer samples diagnosed as Luminal-A tumors and triple negative breast tumors (TNBC). The difference of MTAP expression between fresh tumors and normal tissues was not statistically significant. However, MTAP expression was significantly higher in Luminal-A breast tumors than in TNBC, suggesting the lack of expression in more aggressive breast tumors and the possibility of using the new approaches based on MTAP status in TNBC. PMID:26751376

  17. GnRH increases glucose transporter-1 expression and stimulates glucose uptake in the gonadotroph.

    PubMed

    Harris, Valerie M; Bendre, Sachin V; Gonzalez De Los Santos, Francina; Fite, Alemu; El-Yaman El-Dandachli, Ahmad; Kurenbekova, Lyazat; Abou-Samra, Abdul B; Buggs-Saxton, Colleen

    2012-02-01

    GnRH is the main regulator of the hypothalamic-pituitary-gonadal (H-P-G) axis. GnRH stimulates the pituitary gonadotroph to synthesize and secrete gonadotrophins (LH and FSH), and this effect of GnRH is dependent on the availability of glucose and other nutrients. Little is known about whether GnRH regulates glucose metabolism in the gonadotroph. This study examined the regulation of glucose transporters (Gluts) by GnRH in the LβT2 gonadotroph cell line. Using real-time PCR analysis, the expression of Glut1, -2, -4, and -8 was detected, but Glut1 mRNA expression level was more abundant than the mRNA expression levels of Glut2, -4, and -8. After the treatment of LβT2 cells with GnRH, Glut1 mRNA expression was markedly induced, but there was no GnRH-induction of Glut2, -4, or -8 mRNA expression in LβT2 cells. The effect of GnRH on Glut1 mRNA expression is partly mediated by ERK activation. GnRH increased GLUT1 protein and stimulated GLUT1 translocation to the cell surface of LβT2 cells. Glucose uptake assays were performed in LβT2 cells and showed that GnRH stimulates glucose uptake in the gonadotroph. Finally, exogenous treatment of mice with GnRH increased the expression of Glut1 but not the expression of Glut2, -4, or -8 in the pituitary. Therefore, regulation of glucose metabolism by GnRH via changes in Gluts expression and subcellular location in the pituitary gonadotroph reveals a novel response of the gonadotroph to GnRH.

  18. SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells.

    PubMed

    Wu, Feng; Jordan, Ashley; Kluz, Thomas; Shen, Steven; Sun, Hong; Cartularo, Laura A; Costa, Max

    2016-02-15

    The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study, we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA-mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealed the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. PMID:26780400

  19. [Expression of the apomictic potential and selection for apomixis in sorghum line AS-1a].

    PubMed

    El'konin, L A; Beliaeva, E V; Fadeeva, I Iu

    2012-01-01

    Expression of elements of apomixis was studied for ten seasons in sorghum line AS-la and its backcross hybrids on the 9E and A3 sterile cytoplasms. Cytoembryological analysis revealed aposporous embryo sacks (apo-ESs), their initial cells, and, rare, parthenogeneic proembryos in ovules of line AS-la and its BC2 and BC3 hybrids on the 9E cytoplasm. The A3 sterile cytoplasm suppressed the development of parthenogenetic proembryos, but did not affect the apo-ES formation. The frequency of apomictic elements increased in seasons with high daily temperatures and total precipitation deficiency in the period when the ovule and megagametophyte developed (r = -0.805, P < 0.01). Selection was used to isolate the families where the frequency of ovules with apo-ESs was 28% and the frequency of parthenogenetic proembryos was 14%. Emasculated panicles of line AS-la were pollinated with pollen of line Volzhskoe-4v, which carried the Rs marker dominant gene, responsible for the anthocyan color of coleoptyles and leaves in seedlings. Plants of the maternal type were found in the progenies of these crosses at a frequency of 1.4-28.1%. The genetic structure of the endosperm in grains with maternal-type seedlings was inferred from the electrophoretic patterns of storage proteins (kafirins). The kafirin spectra of grains producing maternal-type seedlings was similar to the spectrum of line AS-la and differed from the spectra of grains producing hybrid seedlings, indicating that the endosperm developed independently when apomictic grains formed in line AS-1a. The results showed that lines with facultative apomixis can be constructed in functionally diploid plants.

  20. Weight gain increases human aromatase expression in mammary gland.

    PubMed

    Chen, Dong; Zhao, Hong; Coon, John S; Ono, Masanori; Pearson, Elizabeth K; Bulun, Serdar E

    2012-05-15

    Adulthood weight gain predicts estrogen receptor-positive breast cancer. Because local estrogen excess in the breast likely contributes to cancer development, and aromatase is the key enzyme in estrogen biosynthesis, we investigated the role of local aromatase expression in weight gain-associated breast cancer risk in a humanized aromatase (Arom(hum)) mouse model containing the coding region and the 5'-regulatory region of the human aromatase gene. Compared with littermates on normal chow, female Arom(hum) mice on a high fat diet gained more weight, and had a larger mammary gland mass with elevated total human aromatase mRNA levels via promoters I.4 and II associated with increased levels of their regulators TNFα and C/EBPβ. There was no difference in total human aromatase mRNA levels in gonadal white adipose tissue. Our data suggest that diet-induced weight gain preferentially stimulates local aromatase expression in the breast, which may lead to local estrogen excess and breast cancer risk.

  1. Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

    SciTech Connect

    Nemoto, Kiyomitsu; Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki; Fujiwara, Hironori; Yokosuka, Akihito; Mimaki, Yoshihiro; Ohizumi, Yasushi; Degawa, Masakuni

    2013-02-15

    Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and

  2. Increased interleukin-6 expression is associated with poor prognosis and acquired cisplatin resistance in head and neck squamous cell carcinoma

    PubMed Central

    GAO, JIAN; ZHAO, SEN; HALSTENSEN, TROND S.

    2016-01-01

    Increased expression of interleukin 6 (IL-6) is associated with poor prognosis and chemoresistance in many different carcinomas, but its role in head and neck squamous cell carcinoma (HNSCC) is still unsettled. Analyzing tumorous mRNA expression data from 399 HNSCC patients revealed that high IL-6 expression predicted poor prognosis. Similar tendency was observed in platinum treated patients, suggesting an IL-6 associated cisplatin resistance. IL-6 increase was also found in two in-house acquired cisplatin-resistant HNSCC cell lines (both basaloid and conventional squamous cell carcinoma) by using microarray analysis. However, although the in-house acquired cisplatin-resistant cell lines had higher basal and markedly increased cisplatin-induced IL-6 expression, IL-6 did not mediate the cisplatin resistance as neither exogenous IL-6 nor IL-6R/gp130 inhibitors affected cisplatin sensitivity. Moreover, the IL-6/STAT3 pathway was impaired in the resistant cell lines, partly due to decreased IL-6R expression. Thus, high IL-6 expression correlated to poor prognosis and acquired cisplatin resistance, but it did not mediate cisplatin resistance in the HNSCC cell lines. PMID:27108527

  3. Carnitine Palmitoyltransferase 1 Increases Lipolysis, UCP1 Protein Expression and Mitochondrial Activity in Brown Adipocytes

    PubMed Central

    Calderon-Dominguez, María; Sebastián, David; Fucho, Raquel; Weber, Minéia; Mir, Joan F.; García-Casarrubios, Ester; Obregón, María Jesús; Zorzano, Antonio; Valverde, Ángela M.; Serra, Dolors

    2016-01-01

    The discovery of active brown adipose tissue (BAT) in adult humans and the fact that it is reduced in obese and diabetic patients have put a spotlight on this tissue as a key player in obesity-induced metabolic disorders. BAT regulates energy expenditure through thermogenesis; therefore, harnessing its thermogenic fat-burning power is an attractive therapeutic approach. We aimed to enhance BAT thermogenesis by increasing its fatty acid oxidation (FAO) rate. Thus, we expressed carnitine palmitoyltransferase 1AM (CPT1AM), a permanently active mutant form of CPT1A (the rate-limiting enzyme in FAO), in a rat brown adipocyte (rBA) cell line through adenoviral infection. We found that CPT1AM-expressing rBA have increased FAO, lipolysis, UCP1 protein levels and mitochondrial activity. Additionally, enhanced FAO reduced the palmitate-induced increase in triglyceride content and the expression of obese and inflammatory markers. Thus, CPT1AM-expressing rBA had enhanced fat-burning capacity and improved lipid-induced derangements. This indicates that CPT1AM-mediated increase in brown adipocytes FAO may be a new approach to the treatment of obesity-induced disorders. PMID:27438137

  4. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain. PMID:7852311

  5. Carnitine Palmitoyltransferase 1 Increases Lipolysis, UCP1 Protein Expression and Mitochondrial Activity in Brown Adipocytes.

    PubMed

    Calderon-Dominguez, María; Sebastián, David; Fucho, Raquel; Weber, Minéia; Mir, Joan F; García-Casarrubios, Ester; Obregón, María Jesús; Zorzano, Antonio; Valverde, Ángela M; Serra, Dolors; Herrero, Laura

    2016-01-01

    The discovery of active brown adipose tissue (BAT) in adult humans and the fact that it is reduced in obese and diabetic patients have put a spotlight on this tissue as a key player in obesity-induced metabolic disorders. BAT regulates energy expenditure through thermogenesis; therefore, harnessing its thermogenic fat-burning power is an attractive therapeutic approach. We aimed to enhance BAT thermogenesis by increasing its fatty acid oxidation (FAO) rate. Thus, we expressed carnitine palmitoyltransferase 1AM (CPT1AM), a permanently active mutant form of CPT1A (the rate-limiting enzyme in FAO), in a rat brown adipocyte (rBA) cell line through adenoviral infection. We found that CPT1AM-expressing rBA have increased FAO, lipolysis, UCP1 protein levels and mitochondrial activity. Additionally, enhanced FAO reduced the palmitate-induced increase in triglyceride content and the expression of obese and inflammatory markers. Thus, CPT1AM-expressing rBA had enhanced fat-burning capacity and improved lipid-induced derangements. This indicates that CPT1AM-mediated increase in brown adipocytes FAO may be a new approach to the treatment of obesity-induced disorders. PMID:27438137

  6. miR-17 inhibitor suppressed osteosarcoma tumor growth and metastasis via increasing PTEN expression

    SciTech Connect

    Gao, Yong; Luo, Ling-hui; Li, Shuai; Yang, Cao

    2014-02-07

    Highlights: • miR-17 was increased in OS tissues and cell lines. • Inhibition of miR-17 suppressed OS cell proliferation. • Inhibition of miR-17 suppressed OS cell migration and invasion. • PTEN was a target of miR-17. • miR-17 was negatively correlated with PTEN in OS tissues. - Abstract: MicroRNAs (miRNAs) play essential roles in cancer development and progression. Here, we investigated the role of miR-17 in the progression and metastasis of osteosarcoma (OS). miR-17 was frequently increased in OS tissues and cell lines. Inhibition of miR-17 in OS cell lines substantially suppressed cell proliferation, migration, and invasion. Phosphatase and tensin homolog (PTEN) was identified as a target of miR-17, and ectopic expression of miR-17 inhibited PTEN by direct binding to its 3′-untranslated region (3′-UTR). Expression of miR-17 was negatively correlated with PTEN in OS tissues. Together, these findings indicate that miR-17 acts as an oncogenic miRNA and may contribute to the progression and metastasis of OS, suggesting miR-17 as a potential novel diagnostic and therapeutic target of OS.

  7. Higher expression and activity of metalloproteinases in human cervical carcinoma cell lines is associated with HPV presence.

    PubMed

    da Silva Cardeal, Laura Beatriz; Brohem, Carla Abdo; Corrêa, Tatiana Caroline Silveira; Winnischofer, Sheila Maria Brochado; Nakano, Fabio; Boccardo, Enrique; Villa, Luisa Lina; Sogayar, Mari Cleide; Maria-Engler, Silvya Stuchi

    2006-10-01

    Matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in 3 human invasive cervical carcinoma cell lines. Two HPV16-positive cell lines (SiHa and CaSki) and an HPV-negative cell line (C33A) were cultured either onto a type-I collagen gel, Matrigel, or plastic, to recreate their three-dimensional growth environment and evaluate the expression of these genes using quantitative real-time PCR. We also analyzed the gelatinolytic activity of MMP-2 and MMP-9 by zymography. We found that HPV (human papillomavirus)-positive cell lines express higher levels of MMP-2, MT1-MMP, and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with higher pro-MMP-2 activity. Furthermore, Matrigel substrate influences MMP-2 expression in both SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP-2, MT1-MMP, and TIMP-2, and that pro-MMP-2 activity is higher in HPV-positive than in HPV-negative cells.

  8. Increased Growth of a Newly Established Mouse Epithelial Cell Line Transformed with HPV-16 E7 in Diabetic Mice

    PubMed Central

    He, Lan; Law, Priscilla T. Y.; Boon, Siaw Shi; Zhang, Chuqing; Ho, Wendy C. S.; Banks, Lawrence; Wong, C. K.; Chan, Juliana C. N.; Chan, Paul K. S.

    2016-01-01

    Epidemiological evidence supports that infection with high-risk types of human papillomavirus (HPV) can interact with host and environmental risk factors to contribute to the development of cervical, oropharyngeal, and other anogenital cancers. In this study, we established a mouse epithelial cancer cell line, designated as Chinese University Papillomavirus-1 (CUP-1), from C57BL/KsJ mice through persistent expression of HPV-16 E7 oncogene. After continuous culturing of up to 200 days with over 60 passages, we showed that CUP-1 became an immortalized and transformed epithelial cell line with continuous E7 expression and persistent reduction of retinoblastoma protein (a known target of E7). This model allowed in-vivo study of interaction between HPV and co-factors of tumorigenesis in syngeneic mice. Diabetes has been shown to increase HPV pathogenicity in different pathological context. Herein, with this newly-established cell line, we uncovered that diabetes promoted CUP-1 xenograft growth in syngeneic db/db mice. In sum, we successfully established a HPV-16 E7 transformed mouse epithelial cell line, which allowed subsequent studies of co-factors in multistep HPV carcinogenesis in an immunocompetent host. More importantly, this study is the very first to demonstrate the promoting effect of diabetes on HPV-associated carcinogenesis in vivo, implicating the importance of cancer surveillance in diabetic environment. PMID:27749912

  9. [Flavonoids contents and expression analysis of related genes in red cell line of Saussurea medusa].

    PubMed

    Wang, Yajie; Li, Houhua; Fu, Wanyi; Gao, Yan; Wang, Bingjie; Li, Ling

    2014-08-01

    Saussurea medusa is a rare traditional Chinese medicinal herb. Besides anti-inflammatory and analgesic activities, it has effects of disinhibiting cold, dispelling dampness and promoting blood circulation. Flavonoids are the main medicinal compounds in S. medusa. Contents of flavonoids and expression of flavonoids biosynthesis related genes in white and red (induced by low temperature, high sucrose and high light) callus were analyzed. The results showed that the total flavone in red line was 3.60 times higher compared to white line. The accumulation of rutin in red line (0.25% of dry weight) was 2.40 times higher compared to white line. Anthocyanins were abundant in red line, with the contents of cyanidin 3-O-glucosidechloride and cyanidin 3-O-succinyl glycoside 0.12% and 0.19% of dry weight respectively. CHS, F3'H, FNS, FLS, DFR and ANS genes were highly expressed in red line compared to white line. Expression of three transcription factors (MYB, bHLH and WD40) in red line was significantly higher than that in white line, especially the expression of MYB (19.70 times higher compared to white line). These results indicated that high expression levels of transcription factors induced high expression of structural genes in red line, thereby enhancing the flavonoids biosynthesis. The expression of bHLH and WD40 was similar, whereas it was significantly different from that of MYB, indicating that bHLH and WD40 could form a binary complex to regulate expression of structural genes and flavonoids biosynthesis. PMID:25423752

  10. [Flavonoids contents and expression analysis of related genes in red cell line of Saussurea medusa].

    PubMed

    Wang, Yajie; Li, Houhua; Fu, Wanyi; Gao, Yan; Wang, Bingjie; Li, Ling

    2014-08-01

    Saussurea medusa is a rare traditional Chinese medicinal herb. Besides anti-inflammatory and analgesic activities, it has effects of disinhibiting cold, dispelling dampness and promoting blood circulation. Flavonoids are the main medicinal compounds in S. medusa. Contents of flavonoids and expression of flavonoids biosynthesis related genes in white and red (induced by low temperature, high sucrose and high light) callus were analyzed. The results showed that the total flavone in red line was 3.60 times higher compared to white line. The accumulation of rutin in red line (0.25% of dry weight) was 2.40 times higher compared to white line. Anthocyanins were abundant in red line, with the contents of cyanidin 3-O-glucosidechloride and cyanidin 3-O-succinyl glycoside 0.12% and 0.19% of dry weight respectively. CHS, F3'H, FNS, FLS, DFR and ANS genes were highly expressed in red line compared to white line. Expression of three transcription factors (MYB, bHLH and WD40) in red line was significantly higher than that in white line, especially the expression of MYB (19.70 times higher compared to white line). These results indicated that high expression levels of transcription factors induced high expression of structural genes in red line, thereby enhancing the flavonoids biosynthesis. The expression of bHLH and WD40 was similar, whereas it was significantly different from that of MYB, indicating that bHLH and WD40 could form a binary complex to regulate expression of structural genes and flavonoids biosynthesis. PMID:25507475

  11. Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines.

    PubMed

    Nemoto, Kiyomitsu; Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki; Fujiwara, Hironori; Yokosuka, Akihito; Mimaki, Yoshihiro; Ohizumi, Yasushi; Degawa, Masakuni

    2013-02-15

    Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines - 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells - were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and suppressions of oxidative stress and cell proliferation.

  12. Heterologous expression of the lipid transfer protein CERT increases therapeutic protein productivity of mammalian cells.

    PubMed

    Florin, Lore; Pegel, Antje; Becker, Eric; Hausser, Angelika; Olayioye, Monilola A; Kaufmann, Hitto

    2009-04-20

    Recent studies have demonstrated that the introduction of transgenes regulating protein transport or affecting post-translational modifications can further improve industrial processes for the production of therapeutic proteins in mammalian cells. Our study on improving therapeutic protein production in CHO cells by heterologous expression of the ceramide transfer protein (CERT) was initiated by the recent discovery that CERT is involved in protein kinase D (PKD)-dependent protein transport from the Golgi to the plasma membrane. We generated a set of CHO DG44 cell lines by stable integration of constructs expressing either CERT wild-type or CERT S132A, a mutant conferring increased lipid transfer activity, or a mock plasmid. CHO cells expressing heterologous CERT demonstrated significantly higher specific productivities of the therapeutic protein HSA when grown in inoculum suspension cultures. This effect translated into significantly increased overall HSA titers in a fed-batch format where cells are grown in chemically defined serum-free media. Furthermore, we could show that CERT also enhanced monoclonal antibody secretion in two IgG production cell lines with different basal productivities. The data demonstrate the potential of CERT engineering to improve mammalian cell culture production processes to yield high amounts of a therapeutic protein product of desired quality. To our knowledge, this is the first study showing a bottle neck in recombinant protein secretion at the Golgi complex in mammalian cells. PMID:19428735

  13. Prostate specific antigen gene expression in androgen insensitive prostate carcinoma subculture cell line.

    PubMed

    Tsui, Ke-Hung; Feng, Tsui-Hsia; Chung, Li-Chuan; Chao, Chun-Hsiang; Chang, Phei-Lang; Juang, Horng-Heng

    2008-01-01

    A novel prostate cancer cell line (PC-J) was isolated from an androgen independent non-prostate specific antigen (non-PSA) producing carcinoma cell line. The homologous correlation between PC-J and PC-3 was determined by short tandem repeat analysis. The PSA promoter activity was detected by transient expression assay in the PC-J and LNCaP cells but not in androgen insensitive PC-3 cells. When the PC-J cells were cotransfected with androgen receptor, androgen receptor coactivators and PSA reporter vector cells, the reporter assays indicated that nuclear receptor coactivator 4 (NCOA4) but not androgen receptor activator 24 (ARA24) increased the sensitivity and maximum stimulation of dihydrotestosterone (DHT)-inducing PSA promoter activity. The RT-PCR assays revealed that the expression of several tumor markers, including interleukin-6, prostate stem cell antigen (PSCA), prostate epithelium-specific Ets transcription factor (PDEF) and matriptase, was lower in the PC-J cells than in the PC-3 cells. This cell model elucidated the regulation of PSA expression and enabled comparison of the gene profile at different stages of metastasis in prostatic carcinoma.

  14. Carotenoid accumulation and carotenogenic gene expression during fruit development in novel interspecific inbred squash lines and their parents.

    PubMed

    Nakkanong, Korakot; Yang, Jing Hua; Zhang, Ming Fang

    2012-06-13

    Carotenoid levels and composition during squash fruit development were compared in Cucurbita moschata , Cucurbita maxima , and two lines of their interspecific inbred lines, namely, Maxchata1 and Maxchata2. Eight genes associated with carotenoid biosynthesis were analyzed by quantitative RT-PCR. The two squash species and their interspecific inbred lines exhibited different qualitative and quantitative carotenoid profiles and regulatory mechanisms. C. moschata had the lowest total carotenoid content and mainly accumulated α-carotene and β-carotene, as expected in a fruit with pale-orange flesh. Low carotenoid content in this species was probably due to the comparatively low expression of all genes investigated, especially PSY1 gene, compared to the other squashes. The predominant carotenoids in C. maxima were violaxanthin and lutein, which produced a corresponding yellow flesh color in mature fruit. The relationship between the expression of the CHYB and ZEP genes may result in almost equal concentrations of violaxanthin and lutein in C. maxima at fruit ripening. In contrast, their interspecific inbred lines principally accumulated lutein and β-carotene, leading to orange flesh color. The PSY1 gene exhibited higher expression levels at earlier stages of fruit development in the Maxchata lines, potentially triggering the increased carotenoid accumulation seen in these fruits. Likewise, the higher transcription level of CHYB gene observed in the two interspecific inbred lines might be correlated with high lutein in these hybrids. However, this study could not explain the observed β-carotene accumulation on the basis of gene expression.

  15. Expressing gait-line symmetry in able-bodied gait

    PubMed Central

    Jeleń, Piotr; Wit, Andrzej; Dudziński, Krzysztof; Nolan, Lee

    2008-01-01

    Background Gait-lines, or the co-ordinates of the progression of the point of application of the vertical ground reaction force, are a commonly reported parameter in most in-sole measuring systems. However, little is known about what is considered a "normal" or "abnormal" gait-line pattern or level of asymmetry. Furthermore, no reference databases on healthy young populations are available for this parameter. Thus the aim of this study is to provide such reference data in order to allow this tool to be better used in gait analysis. Methods Vertical ground reaction force data during several continuous gait cycles were collected using a Computer Dyno Graphy in-sole system® for 77 healthy young able-bodied subjects. A curve (termed gait-line) was obtained from the co-ordinates of the progression of the point of application of the force. An Asymmetry Coefficient Curve (AsC) was calculated between the mean gait-lines for the left and right foot for each subject. AsC limits of ± 1.96 and 3 standard deviations (SD) from the mean were then calculated. Gait-line data from 5 individual subjects displaying pathological gait due to disorders relating to the discopathy of the lumbar spine (three with considerable plantarflexor weakness, two with considerable dorsiflexor weakness) were compared to the AsC results from the able-bodied group. Results The ± 1.96 SD limit suggested that non-pathological gait falls within 12–16% asymmetry for gait-lines. Those exhibiting pathological gait fell outside both the ± 1.96 and ± 3SD limits at several points during stance. The subjects exhibiting considerable plantarflexor weakness all fell outside the ± 1.96SD limit from 30–50% of foot length to toe-off while those exhibiting considerable dorsiflexor weakness fell outside the ± 1.96SD limit between initial contact to 25–40% of foot length, and then surpassed the ± 3SD limit after 55–80% of foot length. Conclusion This analysis of gait-line asymmetry provides a reference

  16. Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway

    SciTech Connect

    Kim, Chae E.; Lee, Seung J.; Seo, Kyo W.; Park, Hye M.; Yun, Jung W.; Bae, Jin U.; Bae, Sun S.; Kim, Chi D.

    2010-05-15

    Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B{sub 4} (LTB{sub 4}) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB{sub 4} production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK among these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB{sub 4}. Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB{sub 4}, subsequent MMP-9 production and plaque rupture.

  17. Erythropoietin (EPO) increases myelin gene expression in CG4 oligodendrocyte cells through the classical EPO receptor.

    PubMed

    Cervellini, Ilaria; Annenkov, Alexander; Brenton, Thomas; Chernajovsky, Yuti; Ghezzi, Pietro; Mengozzi, Manuela

    2013-08-28

    Erythropoietin (EPO) has protective effects in neurodegenerative and neuroinflammatory diseases, including in animal models of multiple sclerosis, where EPO decreases disease severity. EPO also promotes neurogenesis and is protective in models of toxic demyelination. In this study, we asked whether EPO could promote neurorepair by also inducing remyelination. In addition, we investigated whether the effect of EPO could be mediated by the classical erythropoietic EPO receptor (EPOR), since it is still questioned if EPOR is functional in nonhematopoietic cells. Using CG4 cells, a line of rat oligodendrocyte precursor cells, we found that EPO increases the expression of myelin genes (myelin oligodendrocyte glycoprotein [MOG] and myelin basic protein [MBP]). EPO had no effect in wild-type CG4 cells, which do not express EPOR, whereas it increased MOG and MBP expression in cells engineered to overexpress EPOR (CG4-EPOR). This was reflected in a marked increase in MOG protein levels, as detected by Western blot. In these cells, EPO induced by 10-fold the early growth response gene 2 (Egr2), which is required for peripheral myelination. However, Egr2 silencing with a siRNA did not reverse the effect of EPO, indicating that EPO acts through other pathways. In conclusion, EPO induces the expression of myelin genes in oligodendrocytes and this effect requires the presence of EPOR. This study demonstrates that EPOR can mediate neuroreparative effects.

  18. Mu opioid receptor expression is increased in inflammatory bowel diseases: implications for homeostatic intestinal inflammation

    PubMed Central

    Philippe, D; Chakass, D; Thuru, X; Zerbib, P; Tsicopoulos, A; Geboes, K; Bulois, P; Breisse, M; Vorng, H; Gay, J; Colombel, J‐F; Desreumaux, P; Chamaillard, M

    2006-01-01

    Background and aims Recent studies with μ opioid receptor (MOR) deficient mice support a physiological anti‐inflammatory effect of MOR at the colon interface. To better understand the potential pharmacological effect of certain opiates in inflammatory bowel diseases (IBD), we (1) evaluated the regulation in vivo and in vitro of human MOR expression by inflammation; and (2) tested the potential anti‐inflammatory function of a specific opiate (DALDA) in inflamed and resting human mucosa. Patients and methods Expression of MOR mRNA and protein was evaluated in healthy and inflamed small bowel and colonic tissues, isolated peripheral blood mononuclear cells and purified monocytes, and CD4+ and CD8+ T cells from healthy donors and IBD patients. The effect of cytokines and nuclear factor κB (NFκB) activation on MOR expression in lymphocyte T and monocytic human cell lines was assessed. Finally, DALDA induced anti‐inflammatory effect was investigated in mucosal explants from controls and IBD patients. Results MOR was expressed in ileal and colonic enteric neurones as well as in immunocytes such as myeloid cells and CD4+ and CD8+ T cells. Overexpressed in active IBD mucosa, MOR was significantly enhanced by cytokines and repressed by NFκB inhibitor in myeloid and lymphocytic cell lines. Furthermore, ex vivo DALDA treatment dampened tumour necrosis factor α mRNA expression in the colon of active IBD patients. Conclusions Given the increased expression of MOR and the ex vivo beneficial effect of DALDA in active IBD, natural and/or synthetic opioid agonists could help to prevent overt pathological intestinal inflammation. PMID:16299031

  19. Novel cell lines derived from transgenic mice expressing recombinant human proteins. Transgenic hepatoma-derived cell lines.

    PubMed

    Perraud, F; Dalemans, W; Ali-Hadji, D; Pavirani, A

    1992-01-01

    We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human alpha 1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of an onc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human alpha 1-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing. PMID:1369183

  20. Microarray-based detection and expression analysis of extracellular matrix proteins in drug‑resistant ovarian cancer cell lines.

    PubMed

    Januchowski, Radosław; Zawierucha, Piotr; Ruciński, Marcin; Zabel, Maciej

    2014-11-01

    Ovarian cancer is the most lethal gynecological malignancy. Multiple drug resistance (MDR) development leads to resistance of cancer cells to chemotherapy. Microarray methods can provide information regarding new candidate genes that can play a role in resistance to cytostatic drugs. Extracellular matrix (ECM) can influence drug resistance by inhibiting the penetration of the drug into cancer tissue as well as increased apoptosis resistance. In the present study, we report changes in the ECM and related gene expression pattern in methotrexate-, cisplatin-, doxorubicin-, vincristine-, topotecan- and paclitaxel-resistant variants of the W1 ovarian cancer cell line. The resistant variants of the W1 cell line were generated by stepwise selection of cells with an increasing concentration of the indicated drugs. Affymetrix GeneChip® Human Genome U219 Array Strips were used for hybridizations. Independent t-tests were used to determinate the statistical significance of results. Genes whose expression levels were higher than the assumed threshold (upregulated, >5-fold and downregulated, <5-fold) were visualized using the scatter plot method, selected and listed in the tables. Among the investigated genes, expression of 24 genes increased, expression of 14 genes decreased and expression of three genes increased or decreased depending on the cell line. Among the increased genes, expression of 10 increased very significantly, >20-fold. These genes were: ITGB1BP3, COL3A1, COL5A2, COL15A1, TGFBI, DCN, LUM, MATN2, POSTN and EGFL6. The expression of seven genes decreased very significantly: ITGA1, COL1A2, LAMA2, GPC3, KRT23, VIT and HMCN1. The expression pattern of ECM and related genes provided the preliminary view into the role of ECM components in cytostatic drug resistance of cancer cells. The exact role of the investigated genes in drug resistance requires further investigation.

  1. Microarray-based detection and expression analysis of extracellular matrix proteins in drug‑resistant ovarian cancer cell lines.

    PubMed

    Januchowski, Radosław; Zawierucha, Piotr; Ruciński, Marcin; Zabel, Maciej

    2014-11-01

    Ovarian cancer is the most lethal gynecological malignancy. Multiple drug resistance (MDR) development leads to resistance of cancer cells to chemotherapy. Microarray methods can provide information regarding new candidate genes that can play a role in resistance to cytostatic drugs. Extracellular matrix (ECM) can influence drug resistance by inhibiting the penetration of the drug into cancer tissue as well as increased apoptosis resistance. In the present study, we report changes in the ECM and related gene expression pattern in methotrexate-, cisplatin-, doxorubicin-, vincristine-, topotecan- and paclitaxel-resistant variants of the W1 ovarian cancer cell line. The resistant variants of the W1 cell line were generated by stepwise selection of cells with an increasing concentration of the indicated drugs. Affymetrix GeneChip® Human Genome U219 Array Strips were used for hybridizations. Independent t-tests were used to determinate the statistical significance of results. Genes whose expression levels were higher than the assumed threshold (upregulated, >5-fold and downregulated, <5-fold) were visualized using the scatter plot method, selected and listed in the tables. Among the investigated genes, expression of 24 genes increased, expression of 14 genes decreased and expression of three genes increased or decreased depending on the cell line. Among the increased genes, expression of 10 increased very significantly, >20-fold. These genes were: ITGB1BP3, COL3A1, COL5A2, COL15A1, TGFBI, DCN, LUM, MATN2, POSTN and EGFL6. The expression of seven genes decreased very significantly: ITGA1, COL1A2, LAMA2, GPC3, KRT23, VIT and HMCN1. The expression pattern of ECM and related genes provided the preliminary view into the role of ECM components in cytostatic drug resistance of cancer cells. The exact role of the investigated genes in drug resistance requires further investigation. PMID:25199881

  2. Inhibition of hypothalamic MCT1 expression increases food intake and alters orexigenic and anorexigenic neuropeptide expression

    PubMed Central

    Elizondo-Vega, Roberto; Cortés-Campos, Christian; Barahona, María José; Carril, Claudio; Ordenes, Patricio; Salgado, Magdiel; Oyarce, Karina; García-Robles, María de los Angeles

    2016-01-01

    Hypothalamic glucosensing, which involves the detection of glucose concentration changes by brain cells and subsequent release of orexigenic or anorexigenic neuropeptides, is a crucial process that regulates feeding behavior. Arcuate nucleus (AN) neurons are classically thought to be responsible for hypothalamic glucosensing through a direct sensing mechanism; however, recent data has shown a metabolic interaction between tanycytes and AN neurons through lactate that may also be contributing to this process. Monocarboxylate transporter 1 (MCT1) is the main isoform expressed by tanycytes, which could facilitate lactate release to hypothalamic AN neurons. We hypothesize that MCT1 inhibition could alter the metabolic coupling between tanycytes and AN neurons, altering feeding behavior. To test this, we inhibited MCT1 expression using adenovirus-mediated transfection of a shRNA into the third ventricle, transducing ependymal wall cells and tanycytes. Neuropeptide expression and feeding behavior were measured in MCT1-inhibited animals after intracerebroventricular glucose administration following a fasting period. Results showed a loss in glucose regulation of orexigenic neuropeptides and an abnormal expression of anorexigenic neuropeptides in response to fasting. This was accompanied by an increase in food intake and in body weight gain. Taken together, these results indicate that MCT1 expression in tanycytes plays a role in feeding behavior regulation. PMID:27677351

  3. Expression pattern of matrix metalloproteinases in human gynecological cancer cell lines

    PubMed Central

    2010-01-01

    Background Matrix metalloproteinases (MMPs) are involved in the degradation of protein components of the extracellular matrix and thus play an important role in tumor invasion and metastasis. Their expression is related to the progression of gynecological cancers (e.g. endometrial, cervical or ovarian carcinoma). In this study we investigated the expression pattern of the 23 MMPs, currently known in humans, in different gynecological cancer cell lines. Methods In total, cell lines from three endometrium carcinomas (Ishikawa, HEC-1-A, AN3 CA), three cervical carcinomas (HeLa, Caski, SiHa), three chorioncarcinomas (JEG, JAR, BeWo), two ovarian cancers (BG-1, OAW-42) and one teratocarcinoma (PA-1) were examined. The expression of MMPs was analyzed by RT-PCR, Western blot and gelatin zymography. Results We demonstrated that the cell lines examined can constitutively express a wide variety of MMPs on mRNA and protein level. While MMP-2, -11, -14 and -24 were widely expressed, no expression was seen for MMP-12, -16, -20, -25, -26, -27 in any of the cell lines. A broad range of 16 MMPs could be found in the PA1 cells and thus this cell line could be used as a positive control for general MMP experiments. While the three cervical cancer cell lines expressed 10-14 different MMPs, the median expression in endometrial and choriocarcinoma cells was 7 different enzymes. The two investigated ovarian cancer cell lines showed a distinctive difference in the number of expressed MMPs (2 vs. 10). Conclusions Ishikawa, Caski, OAW-42 and BeWo cell lines could be the best choice for all future experiments on MMP regulation and their role in endometrial, cervical, ovarian or choriocarcinoma development, whereas the teratocarcinoma cell line PA1 could be used as a positive control for general MMP experiments. PMID:20942921

  4. Intensity increases of actin layer-lines on activation of the Limulus muscle.

    PubMed Central

    Maéda, Y; Boulin, C; Gabriel, A; Sumner, I; Koch, M H

    1986-01-01

    Small angle x-ray diffraction patterns were recorded from isometrically contracting Limulus (horseshoe crab) telson levator muscle using a multiwire proportional-area detector on the storage ring DORIS. In the pattern a substantial increase in intensity is observed on the thin-filament-associated layer-line at 1/38 nm-1 (the first actin layer-line) with a maximum increase at a radial spacing of R = 0.07 nm-1 but there is a much smaller change in the intensity of the 5.9-nm layer-line, which also arises from the thin filament structure. The results suggest that during contraction the myosin heads, presumably being attached to the thin filaments, are arranged along the long-stranded helical tracks of the thin filaments but that the spatial relationship between the heads and the actin monomers varies. Intensity increases have also been observed (Maéda et al., manuscript in preparation) in the part of the patterns from frog muscle and barnacle muscle, which are attributable to the first actin layer-line. It is thus likely that the intensity increase of the first actin layer-line on the Limulus pattern is associated not with structural features which are special to Limulus muscle, but with the tension generating processes that are shared by muscles in general. Images FIGURE 1 FIGURE 2 PMID:3801566

  5. Constitutive Expression of GATA4 Dramatically Increases the Cardiogenic Potential of D3 Mouse Embryonic Stem Cells

    PubMed Central

    Laemmle, Lillian L.; Cohen, Justus B.; Glorioso, Joseph C.

    2016-01-01

    The transcription factor GATA binding protein 4 (GATA4) is a vital regulator of cardiac programming that acts by inducing the expression of many different genes involved in cardiomyogenesis. Here we generated a D3 mouse embryonic stem cell line that constitutively expresses high levels of GATA4 and show that these cells have dramatically increased cardiogenic potential compared to an eGFP-expressing control cell line. Embryoid bodies (EB) derived from the D3-GATA4 line displayed increased levels of cardiac gene expression and showed more abundant cardiomyocyte differentiation than control eGFP EB. These cells and two additional lines expressing lower levels of GATA4 provide a platform to screen previously untested cardiac genes and gene combinations for their ability to further increase the efficiency of cardiomyocyte differentiation beyond that achieved by transgenic GATA4 alone. Non-integrative delivery of identified gene combinations will aid in the production of differentiated cells for the treatment of ischemic cardiomyopathy. PMID:27441042

  6. Increased expression of S100A4, a metastasis-associated gene, in human colorectal adenocarcinomas.

    PubMed

    Takenaga, K; Nakanishi, H; Wada, K; Suzuki, M; Matsuzaki, O; Matsuura, A; Endo, H

    1997-12-01

    The S100A4 gene (also known as pEL98/mts1/p9Ka/18A2/42A/calvasculin /FSP1/CAPL) encoding an S100-related calcium-binding protein is implied to be involved in the invasion and metastasis of murine tumor cells. In the present study, the expression of S100A4 in human colorectal adenocarcinoma cell lines (SW837, LoVo, DLD-1, HT-29, SW480, SW620, WiDr, and Colo201) and surgically resected neoplastic tissues was examined to investigate whether S100A4 plays a role in the invasion and metastasis of human tumor cells. Northern blot analysis using total RNA isolated from the adenocarcinoma cell lines revealed that five of the eight cell lines expressed substantial amounts of S100A4 mRNA. Normal colon fibroblasts (CCD-18Co) expressed little of the RNA. Using surgically resected specimens, it seemed that the amount of S100A4 mRNA in adenomas was nearly equal to that in normal colonic mucosa, whereas adenocarcinomas expressed a significantly higher amount of the RNA than did the adjacent normal colonic mucosa. Immunohistochemical analysis using formalin-fixed paraffin-embedded surgical specimens and monoclonal anti-S100A4 antibody demonstrated that none of 12 adenoma specimens were immunopositive, whereas 8 of 18 (44%) focal carcinomas in carcinoma in adenoma specimens and 50 of 53 (94%) adenocarcinoma specimens were immunopositive. Interestingly, the incidence of immunopositive cells increased according to the depth of invasion, and nearly all of the carcinoma cells in 14 metastases in the liver were positive. These results suggest that S100A4 may be involved in the progression and the metastatic process of human colorectal neoplastic cells. PMID:9815629

  7. Brazilian propolis extract increases leptin expression in mouse adipocytes.

    PubMed

    Washio, Kohei; Shimamoto, Yoshinori; Kitamura, Hiroshi

    2015-01-01

    We investigated the anti-obesity effects of Brazilian green propolis ethanol extract using a mouse model of obesity. Repeated intraperitoneal injection of propolis (100 mg/kg twice a week) caused feeding suppression in C57BL/6 mice, whereas this treatment had negligible effects on C57BL/6 ob/ob mice. Since C57BL/6 ob/ob mice have a missense mutation in the Lep gene, leptin is likely to contribute to the propolis-induced feeding suppression. We found that propolis treatment indeed clearly increased leptin mRNA production in the visceral adipose tissues. Moreover, propolis extract directly elevated leptin expression in differentiated 3T3-L1 adipocytes. Artepillin C, an important organic compound found in Brazilian green propolis, failed to induce leptin mRNA in 3T3-L1 cells. Compounds other than artepillin C in Brazilian propolis must thus cause leptin induction in adipocytes, possibly resulting in the suppression of feeding and obesity.

  8. BHK cell lines with increased rates of gene amplification are hypersensitive to ultraviolet light

    SciTech Connect

    Giulotto, E.; Bertoni, L.; Attolini, C.; Rainaldi, G.; Anglana, M. )

    1991-04-15

    Four cell lines (MP1, -4, -5, -7), isolated from baby hamster kidney cells after simultaneous selection with N-(phosphonacetyl)-L-aspartate and methotrexate, have previously been shown to amplify their DNA at an increased rate. We now show that all four lines are hypersensitive to killing by UV light and mitomycin C. At high doses of UV light or mitomycin C, the MP lines survived less than 10% or less than 5% as well as parental cells, respectively. After UV irradiation, inhibition of DNA and RNA synthesis was greater in MP than in parental cells, and recovery was slower or absent. A 2- to 3.5-fold increase in the frequency of UV-induced sister chromatid exchange was also seen in the four cell lines. In MP5, unscheduled DNA replication after treatment with UV light was only approximately 70% as great as in parental cells and the other MP lines. In MP4 and MP7 cells S phase was elongated. Although their individual properties confirm that the four cell lines are independent, their common properties suggest a relationship between tolerance of DNA damage and gene amplification.

  9. Snake venom causes apoptosis by increasing the reactive oxygen species in colorectal and breast cancer cell lines

    PubMed Central

    Al-Asmari, Abdulrahman Khazim; Riyasdeen, Anvarbatcha; Al-Shahrani, Mohammad Hamed; Islam, Mozaffarul

    2016-01-01

    Snake venom possesses various kinds of proteins and neurotoxic polypeptides, which can negatively interfere with the neurotransmitter signaling cascade. This phenomenon occurs mainly due to the blocking of ion channels in the body system. Envenomation prevents or severely interrupts nerve impulses from being transmitted, inhibition of adenosine triphosphate synthesis, and proper functioning of the cardiac muscles. However, some beneficial properties of venoms have also been reported. The aim of this study was to examine the snake venom as an anticancer agent due to its inhibitory effects on cancer progression such as cell motility, cell invasion, and colony formation. In this study, the effect of venoms on phenotypic changes and the change on molecular level in colorectal and breast cancer cell lines were examined. A reduction of 60%–90% in cell motility, colony formation, and cell invasion was observed when these cell lines were treated with different concentrations of snake venom. In addition, the increase in oxidative stress that results in an increase in the number of apoptotic cancer cells was significantly higher in the venom-treated cell lines. Further analysis showed that there was a decrease in the expression of pro-inflammatory cytokines and signaling proteins, strongly suggesting a promising role for snake venom against breast and colorectal cancer cell progression. In conclusion, the snake venoms used in this study showed significant anticancer properties against colorectal and breast cancer cell lines. PMID:27799796

  10. FGF23 is elevated in multiple myeloma and increases heparanase expression by tumor cells

    PubMed Central

    Suvannasankha, Attaya; Tompkins, Douglas R.; Edwards, Daniel F.; Petyaykina, Katarina V.; Crean, Colin D.; Fournier, Pierrick G.; Parker, Jamie M.; Sandusky, George E.; Ichikawa, Shoji; Imel, Erik A.; Chirgwin, John M.

    2015-01-01

    Multiply myeloma (MM) grows in and destroys bone, where osteocytes secrete FGF23, a hormone which affects phosphate homeostasis and aging. We report that multiple myeloma (MM) cells express receptors for and respond to FGF23. FGF23 increased mRNA for EGR1 and its target heparanase, a pro-osteolytic factor in MM. FGF23 signals through a complex of klotho and a classical FGF receptor (FGFR); both were expressed by MM cell lines and patient samples. Bone marrow plasma cells from 42 MM patients stained positively for klotho, while plasma cells from 8 patients with monoclonal gammopathy of undetermined significance (MGUS) and 6 controls were negative. Intact, active FGF23 was increased 2.9X in sera of MM patients compared to controls. FGF23 was not expressed by human MM cells, but co-culture with mouse bone increased its mRNA. The FGFR inhibitor NVP-BGJ398 blocked the heparanase response to FGF23. NVP-BGJ398 did not inhibit 8226 growth in vitro but significantly suppressed growth in bone and induction of the osteoclast regulator RANK ligand, while decreasing heparanase mRNA. The bone microenvironment provides resistance to some anti-tumor drugs but increased the activity of NVP-BGJ398 against 8226 cells. The FGF23/klotho/heparanase signaling axis may offer targets for treatment of MM in bone. PMID:25944690

  11. Ectopic expression of new alternative splice variant of Smac/DIABLO increases mammospheres formation

    PubMed Central

    Martinez-Ruiz, Gustavo U; Victoria-Acosta, Georgina; Vazquez-Santillan, Karla I; Jimenez-Hernandez, Luis; Muñoz-Galindo, Laura; Ceballos-Cancino, Gisela; Maldonado, Vilma; Melendez-Zajgla, Jorge

    2014-01-01

    Smac-α is a mitochondrial protein that, during apoptosis, is translocated to the cytoplasm, where it negatively regulates members of the inhibitor of apoptosis (IAP) family via the IAP-binding motif (IBM) contained within its amino-terminus. Here, we describe a new alternative splice variant from Smac gene, which we have named Smac-ε. Smac-ε lacks both an IBM and a mitochondrial-targeting signal (MTS) element. Smac-ε mRNA exhibits a tissue-specific expression pattern in healthy human tissues as well as in several cancer cell lines. The steady-state levels of endogenous Smac-ε protein is regulated by the proteasomal pathway. When ectopically expressed, this isoform presents a cytosolic localization and is unable to associate with or to regulate the expression of X-linked Inhibitor of apoptosis protein, the best-studied member of IAP family. Nevertheless, over-expression of Smac-ε increases mammosphere formation. Whole genome expression analyses from these mammospheres show activation of several pro-survival and growth pathways, including Estrogen-Receptor signaling. In conclusion, our results support the functionality of this new Smac isoform. PMID:25337193

  12. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    SciTech Connect

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  13. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

    SciTech Connect

    Sustarsic, Elahu G.; Junnila, Riia K.; Kopchick, John J.

    2013-11-08

    Highlights: •Most cancer types of the NCI60 have sub-sets of cell lines with high GHR expression. •GHR is highly expressed in melanoma cell lines. •GHR is elevated in advanced stage IV metastatic tumors vs. stage III. •GH treatment of metastatic melanoma cell lines alters growth and cell signaling. -- Abstract: Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on

  14. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression

    PubMed Central

    Prados, Jose; Caba, Octavio; Cabeza, Laura; Berdasco, Maria; Gónzalez, Beatriz; Melguizo, Consolación

    2015-01-01

    Background The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated. Methods Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2’-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed. Results Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines. Conclusions These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma

  15. A reporter mouse line with doxycyclin-inducible expression of β-glucosidase.

    PubMed

    Jay, Freya F; Schneider, Marlon R

    2014-12-01

    Mouse lines allowing the inducible expression of transgenes became essential tools for studying gene function and for developing accurate animal models for human diseases. A key component of this tool is the availability of "reporter" lines, mice expressing transgenes encoding easily detectable enzymes or other proteins normally not associated with eukaryotic tissues. Such lines may be suitable for a number of applications, including lineage tracing, label-retaining experiments, and the identification and monitoring of regulatory elements important for tissue-specific gene expression. However, only a limited number of reporter lines suitable for inducible expression systems are available. Here, we employed pronuclear DNA microinjection to generate a new reporter mouse line that allows the inducible expression of β-glucosidase, a recently reported stable and easily detectable protein, upon administration of doxycyclin to the drinking water. This novel line was established in the widely used inbreed background C57BL/6, and the transgene is transmitted between generations in a Mendelian fashion. When crossed to a K14-rtTA mouse line, activation of β-glucosidase expression in the epidermal basal layer is easily detected in double-transgenic animals receiving doxycyclin, while no expression is seen in double-transgenic mice without doxycyclin treatment or in animals carrying only one transgene. We anticipate that this new mouse line will become a valuable tool for a number of applications in vivo, including label-retaining experiments and testing the appropriate regulation of rtTA cassettes under different promoters in novel transgenic mouse lines. PMID:25091595

  16. A reporter mouse line with doxycyclin-inducible expression of β-glucosidase.

    PubMed

    Jay, Freya F; Schneider, Marlon R

    2014-12-01

    Mouse lines allowing the inducible expression of transgenes became essential tools for studying gene function and for developing accurate animal models for human diseases. A key component of this tool is the availability of "reporter" lines, mice expressing transgenes encoding easily detectable enzymes or other proteins normally not associated with eukaryotic tissues. Such lines may be suitable for a number of applications, including lineage tracing, label-retaining experiments, and the identification and monitoring of regulatory elements important for tissue-specific gene expression. However, only a limited number of reporter lines suitable for inducible expression systems are available. Here, we employed pronuclear DNA microinjection to generate a new reporter mouse line that allows the inducible expression of β-glucosidase, a recently reported stable and easily detectable protein, upon administration of doxycyclin to the drinking water. This novel line was established in the widely used inbreed background C57BL/6, and the transgene is transmitted between generations in a Mendelian fashion. When crossed to a K14-rtTA mouse line, activation of β-glucosidase expression in the epidermal basal layer is easily detected in double-transgenic animals receiving doxycyclin, while no expression is seen in double-transgenic mice without doxycyclin treatment or in animals carrying only one transgene. We anticipate that this new mouse line will become a valuable tool for a number of applications in vivo, including label-retaining experiments and testing the appropriate regulation of rtTA cassettes under different promoters in novel transgenic mouse lines.

  17. Transient HEXA expression in a transformed human fetal Tay-Sachs disease neuroglial cell line

    SciTech Connect

    Fernandes, M.J.; Hechtman, P.; Kaplan, F.

    1994-09-01

    Tay-Sachs disease (TSD) is a severe neurodegenerative disorder characterized by the accumulation of GM{sub 2} ganglioside in the neurons of the central cortex. The recessively inherited disorder results from deficiency of hexosaminidase A (Hex A), a heterodimer of an {alpha} and {beta} subunit encoded by the HEXA and HEXB genes. Expression of HEXA mutations in COS cells has several disadvantages including high endogenous hexosaminidase activity. We report a new transient expression system with very low endogenous Hex A activity. An SV40-transformed fetal TSD neuroglial cell line was assessed for transient expression of the HEXA gene. pCMV{alpha}, a vector incorporating the cytomegalovirus promoter with the human {alpha}-subunit cDNA insert, proved to be the most efficient expression vector. Transfection of 4x10{sup 6} cells with 5-20 {mu}g of plasmid resulted in 100 to 500-fold Hex A activity (4MUGS hydrolysis) relative to mock-transfected cells. Use of pCMV{beta}-Gal as a control for transfection efficiency indicated that 10-20% of cells were transfected. Hex A specific activity increased for at least 72 h post-transfection. This new transient expression system should greatly improve the characterization of mutations in which low levels of HEXA expression result in milder clinical phenotypes and permit studies on enzymatic properties of mutant forms of Hex A. Since the cells used are of CNS origin and synthesize gangliosides, it should also be possible to study, in culture, the metabolic phenotype associated with TSD.

  18. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment.

    PubMed

    Sustarsic, Elahu G; Junnila, Riia K; Kopchick, John J

    2013-11-01

    Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute's NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma. PMID:24134847

  19. Increased sensitivity of multidrug-resistant myeloid leukemia cell lines to lovastatin.

    PubMed

    Maksumova, L; Ohnishi, K; Muratkhodjaev, F; Zhang, W; Pan, L; Takeshita, A; Ohno, R

    2000-08-01

    Lovastatin, a competitive inhibitor of HMG-CoA reductase, reportedly inhibits proliferation and induces apoptosis of tumor cells with MDR-1 coded P-glycoprotein (Pgp) expression. In this study we investigated the sensitivity to lovastatin of eight myeloid leukemia cell lines: K562, NOMO-1, NB4 and its retinoic acid (RA) resistant subline NB4/RA, and their multidrug-resistant (MDR) sublines: K562/ADR, NOMO-1/ADR, NB4/MDR and NB4/RA/MDR. MTT and apoptosis assays revealed that K562/ADR, NOMO-1/ADR and NB4/RA/MDR were more sensitive to lovastatin than their parental cell lines, while NB4/MDR showed the same level of sensitivity as parental NB4 cells, which already were very sensitive to lovastatin. Significant elevation of transcript levels of HMG-CoA reductase was observed by semiquantitative RT-PCR analysis in more than three lovastatin-sensitive MDR sublines, but not in NB4/MDR compared with the parental cell lines. HMG-CoA reductase mRNA levels were up-regulated more than two-fold by the exposure to lovastatin in all of the parental non-Pgp-expressing cell lines. In NB4/MDR, HMG-CoA reductase mRNA level was elevated to a similar extent as in parental NB4, whereas in three other MDR sublines which showed preferential sensitivity to lovastatin, their HMG-CoA reductase mRNA levels were not significantly elevated after 24- and 48-h treatment with lovastatin. These results indicate a connection between drug resistance and regulation of the mevalonate pathway, and further strengthen the clinical possibility that drug resistant leukemias would be susceptible to treatment with lovastatin. PMID:10942241

  20. Differential expression of novH and CTGF in human glioma cell lines

    PubMed Central

    Xin, L W; Martinerie, C; Zumkeller, W; Westphal, M; Perbal, B

    1996-01-01

    Aims—(1) To investigate the expression in human derived glioblastoma cell lines of two structurally related genes, novH (nephroblastoma overexpressed gene) and CTGF (connective tissue growth factor), which encode putative insulin-like growth factor binding proteins of a novel type. (2) To investigate whether the same transcription factors regulate CTGF and novH expression. Methods—Expression of novH and CTGF was analysed in 24 glioblastoma derived cell lines by northern blotting. The CTGF promoter region was characterised by nucleotide sequencing, RNase protection experiments, by transient transfections, and CAT assays. Results—CTGF and novH mRNA levels differed in the glioma cell lines studied. NovH and CTGF genes were not co-expressed in all cell lines. The CTGF promoter region was highly conserved compared with the corresponding region in the mouse (FISP12) and exhibited in vitro transcriptional activity. Conclusions—Although the coding regions of novH and CTGF are highly homologous, their promoter regions are substantially different, suggesting that these two genes may be regulated by different mechanisms. Considering that novH and CTGF are likely to be, respectively, negative and positive regulators of growth and that some glioma cell lines expressing novH are not tumorigenic, expression of these two genes might represent a key element in determining the stage of differentiation or the malignant potential, or both, of some tumour cell lines. Images PMID:16696057

  1. The investigation of miR-221-3p and PAK1 gene expressions in breast cancer cell lines.

    PubMed

    Ergun, Sercan; Tayeb, Tayeb Sadiq; Arslan, Ahmet; Temiz, Ebru; Arman, Kaifee; Safdar, Muhammad; Dağlı, Hasan; Korkmaz, Murat; Nacarkahya, Gülper; Kırkbeş, Sevil; Oztuzcu, Serdar

    2015-01-25

    The most common malignancy in women is breast cancer. Drug resistance in the treatment of cancer still remains a major clinical concern. Resistance to tamoxifen is seen in half of the recurrences in breast cancer. The anti-estrogen tamoxifen gains agonistic property by transactivating ERα. PAK1-mediated phosphorylation of serine 305 (S305) of ERα leads to resistance to tamoxifen. In our study, PAK1-induced suggestive tamoxifen resistance was designed. According to our hypothesis, phosphorylation of ERα-S305 by PAK1 may be reversed by PAK1 transcriptional inhibition by miR-221-3p due to miR-221-3p targeting the 3' UTR of PAK1. For this purpose, we used Real-time PCR (qRT-PCR) to measure the expression level of miR-221-3p in ER-positive breast cancer cell lines (ZR-75-1, MCF7) and breast epithelial cell line, hTERT-HME1, as control in the laboratory in our department. The increase in the expression of PAK1 depending on miR-221-3p may be related to ZR-75-1 cell line which has invasive characteristic but other two ER+ cancer cell lines, MCF7 and HCC1500, have milder cancer severity. miR-221-3p may have a role on regulation of PAK1 expression because miR-221-3p expression level decreases while PAK1 expression level increases in SKBR3 cell line. miR-221-3p and PAK1 expressions in MDA-MB-231 cell line are higher than that of hTERT-HME1 cell line. This may mean that miR-221-3p has no regulatory effect on of PAK1 expression in this cell line. According to these results, miR-221-3p may give crucial information about molecular mechanism of the disease upon PAK1 activity or different mechanisms with respect to histopathology and severity of breast cancer.

  2. Enhanced expression of multidrug resistance-associated protein 2 and reduced expression of aquaglyceroporin 3 in an arsenic-resistant human cell line.

    PubMed

    Lee, Te-Chang; Ho, I-Ching; Lu, Wen-Jen; Huang, Jin-ding

    2006-07-01

    Arsenic-resistant cells (R15), derived from a human lung adenocarcinoma cell line (CL3), were 10-fold more resistant to sodium arsenite (As(III)). Because R15 cells accumulated less arsenic than parental CL3 cells, this arsenic resistance may be due to higher efflux and/or lower uptake of As(III). We therefore compared expression of the multidrug resistance-associated proteins MRP1, MRP2, and MRP3 in these two cell lines. MRP2 expression was 5-fold higher in R15 cells than in CL3 cells, whereas MRP1 and MRP3 expression levels were similar. Furthermore, verapamil and cyclosporin A, inhibitors of multidrug resistance transporters, significantly reduced the efflux of arsenic from R15. Thus, increased arsenic extrusion by MRP2 may contribute to arsenic resistance in R15 cells. We also examined the expression of several aquaglyceroporins (AQPs), which mediate As(III) uptake by cells. Little AQP7 or AQP9 mRNA was detected by reverse transcription-PCR in either cell line, whereas AQP3 mRNA expression was 2-fold lower in R15 cells than in CL3 cells. When AQP3 expression in CL3 cells was knocked down by RNA interference, CL3 cells accumulated less arsenic and became more resistant to As(III). Conversely, overexpression of AQP3 in human embryonic kidney 293T cells increased arsenic accumulation, and the cells were more susceptible to As(III) than 293T cells transfected with vector alone. These results suggest that AQP3 is involved in As(III) accumulation. Taken together, our results suggest that enhanced expression of MRP2 and lower expression of AQP3 are responsible for lower arsenic accumulation in arsenic-resistant R15 cells.

  3. Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation

    PubMed Central

    Chen, Huiyong; Choesang, Tenzin; Li, Huihui; Sun, Shuming; Pham, Petra; Bao, Weili; Feola, Maria; Westerman, Mark; Li, Guiyuan; Follenzi, Antonia; Blanc, Lionel; Rivella, Stefano; Fleming, Robert E.; Ginzburg, Yelena Z.

    2016-01-01

    Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbbth1/th1 (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes. PMID:26635037

  4. Gene expression array analyses predict increased proto-oncogene expression in MMTV induced mammary tumors.

    PubMed

    Popken-Harris, Pamela; Kirchhof, Nicole; Harrison, Ben; Harris, Lester F

    2006-08-01

    Exogenous infection by milk-borne mouse mammary tumor viruses (MMTV) typically induce mouse mammary tumors in genetically susceptible mice at a rate of 90-95% by 1 year of age. In contrast to other transforming retroviruses, MMTV acts as an insertional mutagen and under the influence of steroid hormones induces oncogenic transformation after insertion into the host genome. As these events correspond with increases in adjacent proto-oncogene transcription, we used expression array profiling to determine which commonly associated MMTV insertion site proto-oncogenes were transcriptionally active in MMTV induced mouse mammary tumors. To verify our gene expression array results we developed real-time quantitative RT-PCR assays for the common MMTV insertion site genes found in RIII/Sa mice (int-1/wnt-1, int-2/fgf-3, int-3/Notch 4, and fgf8/AIGF) as well as two genes that were consistently up regulated (CCND1, and MAT-8) and two genes that were consistently down regulated (FN1 and MAT-8) in the MMTV induced tumors as compared to normal mammary gland. Finally, each tumor was also examined histopathologically. Our expression array findings support a model whereby just one or a few common MMTV insertions into the host genome sets up a dominant cascade of events that leave a characteristic molecular signature.

  5. Protein expression profile related to cisplatin resistance in bladder cancer cell lines detected by two-dimensional gel electrophoresis.

    PubMed

    Taoka, Yoshinori; Matsumoto, Kazumasa; Ohashi, Kazuya; Minamida, Satoru; Hagiwara, Masahiro; Nagi, Shoji; Saito, Tatsuya; Kodera, Yoshio; Iwamura, Masatsugu

    2015-01-01

    We used a proteomic approach to compare the differentially regulated protein expression profiles of cisplatin-naïve and cisplatin-resistant bladder cancer cell lines to screen candidate molecules related to cisplatin resistance. The cisplatin-resistant cell line T24 was established by the stepwise exposure of T24 cells to up to 40 μM of cisplatin. We performed a comprehensive study of protein expression in bladder cancer cell lines that included cisplatin-naïve (T24) and cisplatin-resistant cells (T24CDDPR) by means of agarose two-dimensional gel electrophoresis followed by analysis of liquid chromatography tandem mass spectroscopy. We identified 25 obviously different spots for T24 and T24 CDDPR. Seven spots had increased expression and 18 spots had decreased expression in T24CDDPR compared to those in T24. Cytoskeletal proteins and enzyme modulators were prominent among differential proteins. Of the 25 proteins, we selected HNRNPA3, PCK2, PPL, PGK1, TKT, SERPINB2, GOT2, and EIF3A for further validation by Western blot. HNRNPA3, PGK1, TKT, and SERPINB2 had more than 1.5-times incremental expression in T24CDDPR compared to that in T24. PCK2 and PPL expressions were decreased less than 20% in T24CDDPR compared to that in T24. The results of 25 new proteins in this study could be valuable and could lead to the development of a new molecular marker. PMID:26299484

  6. Increased deposition of polychlorinated biphenyls (PCBs) under an AC high-voltage power line

    NASA Astrophysics Data System (ADS)

    Öberg, Tomas; Peltola, Pasi

    2009-12-01

    There is considerable public concern regarding the potential risks to health of electromagnetic fields in general and high-voltage power lines in particular. As epidemiological findings are not supported by a clearly defined mechanism of direct magnetic field interactions with the human body, potential indirect effects are of interest. It has been suggested that an increased exposure to chemical pollutants could occur near high-voltage power lines due to formation and deposition of charged aerosols. The current study reports empirical evidence that seems to support this hypothesis. The deposition of 18 congeners of polychlorinated biphenyls (PCBs) was studied by collecting samples of pine needles under a 400 kV AC power line and at reference sites in the vicinity. Compared to the reference sites, the average deposition of PCB congeners under the power line was almost double. This difference between the two groups of samples was statistically significant. While it is premature to draw any conclusions regarding the human exposure near high-voltage power lines, the issue deserves attention and further investigations.

  7. The expression of S100P increases and promotes cellular proliferation by increasing nuclear translocation of β-catenin in endometrial cancer.

    PubMed

    Guo, Luyan; Chen, Shuqin; Jiang, Hongye; Huang, Jiaming; Jin, Wenyan; Yao, Shuzhong

    2014-01-01

    There is increasing evidence suggesting that S100P has a significant role in cancer, and is associated with poor clinical outcomes. The expression of S100P mRNA and protein in endometrial cancer and normal endometrium tissues was detected by real-time quantitative RT-PCR and immunohistochemistry. Moreover, we reduced the expression of S100P in HEC-1A and Ishikawa endometrial cancer cell lines by siRNA transfection. Based on the reduced S100P mRNA expression, we measured the effects of S100P on cellular proliferation by the cell-counting kit-8. Nuclear β-catenin protein level was detected by western blotting. Cyclin D1 and c-myc mRNA expression regulated by β-catenin was detected by real-time quantitative RT-PCR. We found that the expression of S100P mRNA and protein increased in endometrial cancer tissues compared with the normal endometrium. Local S100P expression progressively increased from pathologic differenciation grade 1 to 3. After reducing the S100P expression, the cellular proliferation ability, nuclear β-catenin protein level, cyclin D1 and c-myc mRNA levels reduced. It indicated that S100P could promote cell proliferation by increasing nuclear translocation of β-catenin. The expression of S100P mRNA and protein in endometrial cancer significantly increased and is associated with pathologic differenciation grade. S100P may promote endometrial cell proliferation by increasing nuclear translocation of β-catenin.

  8. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

    PubMed Central

    Besser, Stefanie; Sicker, Marit; Marx, Grit; Winkler, Ulrike; Eulenburg, Volker; Hülsmann, Swen; Hirrlinger, Johannes

    2015-01-01

    GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type. PMID:26076353

  9. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons.

    PubMed

    Besser, Stefanie; Sicker, Marit; Marx, Grit; Winkler, Ulrike; Eulenburg, Volker; Hülsmann, Swen; Hirrlinger, Johannes

    2015-01-01

    GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type. PMID:26076353

  10. Heparin treatment increases thioredoxin interacting protein expression in hepatocellular carcinoma cells.

    PubMed

    Gunes, Aysim; Iscan, Evin; Topel, Hande; Avci, Sanem Tercan; Gumustekin, Mukaddes; Erdal, Esra; Atabey, Nese

    2015-08-01

    Heparins play an important role in cell growth, differentiation, migration and invasion. However, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. To determine the effect of heparin on gene expression, we performed a cDNA microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. In this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. We determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. We showed the importance of heparin mediated histone modifications and down-regulation of Enhancer of zeste 2 polycomb repressive complex 2 expression for heparin mediated overexpression of thioredoxin-interacting protein. When we tested biological significance of these data; we observed that cells overexpressing thioredoxin-interacting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. Interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. In conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. This study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis.

  11. Regret Expression and Social Learning Increases Delay to Sexual Gratification

    PubMed Central

    Quisenberry, Amanda J.; Eddy, Celia R.; Patterson, David L.; Franck, Christopher T.; Bickel, Warren K.

    2015-01-01

    Objective Modification and prevention of risky sexual behavior is important to individuals’ health and public health policy. This study employed a novel sexual discounting task to elucidate the effects of social learning and regret expression on delay to sexual gratification in a behavioral task. Methods Amazon Mechanical Turk Workers were assigned to hear one of three scenarios about a friend who engages in similar sexual behavior. The scenarios included a positive health consequence, a negative health consequence or a negative health consequence with the expression of regret. After reading one scenario, participants were asked to select from 60 images, those with whom they would have casual sex. Of the selected images, participants chose one image each for the person they most and least want to have sex with and person most and least likely to have a sexually transmitted infection. They then answered questions about engaging in unprotected sex now or waiting some delay for condom-protected sex in each partner condition. Results Results indicate that the negative health outcome scenario with regret expression resulted in delayed sexual gratification in the most attractive and least STI partner conditions, whereas in the least attractive and most STI partner conditions the negative health outcome with and without regret resulted in delayed sexual gratification. Conclusions Results suggest that the sexual discounting task is a relevant laboratory measure and the framing of information to include regret expression may be relevant for prevention of risky sexual behavior. PMID:26280349

  12. Contact sensitizers specifically increase MHC class II expression on murine immature dendritic cells.

    PubMed

    Herouet, C; Cottin, M; LeClaire, J; Enk, A; Rousset, F

    2000-01-01

    Contact sensitivity is a T-cell-mediated immune disease that can occur when low-molecular-weight chemicals penetrate the skin. In vivo topical application of chemical sensitizers results in morphological modification of Langerhans cells (LC). Moreover, within 18 h, LC increase their major histocompatibility complex (MHC) class II antigens expression and migrate to lymph nodes where they present the sensitizer to T lymphocytes. We wanted to determine if such an effect could also be observed in vitro. However, because of the high genetic diversity encountered in humans, assays were performed with dendritic cells (DC) obtained from a Balb/c mouse strain. The capacity of a strong sensitizer, DNBS (2,4-dinitrobenzene sulfonic acid), to modulate the phenotype of bone marrow-derived DC in vitro, was investigated. A specific and marked increase of MHC class II molecules expression was observed within 18 h. To eliminate the use of animals in sensitization studies, the XS52 DC line was tested at an immature stage. A 30-min contact with the strong sensitizers DNBS and oxazolone, or the moderate mercaptobenzothiazole, resulted in upregulation of MHC class II molecules expression, analyzed after 18-h incubation. This effect was not observed with irritants (dimethyl sulfoxide and sodium lauryl sulfate) nor with a neutral molecule (sodium chloride). These data suggested the possibility of developing an in vitro model for the identification of the sensitizing potential of chemicals, using a constant and non animal-consuming material.

  13. HEK293 cell line: a vehicle for the expression of recombinant proteins.

    PubMed

    Thomas, Philip; Smart, Trevor G

    2005-01-01

    The HEK cell line has been extensively used as an expression tool for recombinant proteins since it was generated over 25 years ago. Although of epithelial origin, its biochemical machinery is capable of carrying out most of the post-translational folding and processing required to generate functional, mature protein from a wide spectrum of both mammalian and non-mammalian nucleic acids. Though popular as a transient expression system, this cell type has also seen wide use in stably transfected forms (i.e. transformed cells) to study a variety of cell-biological questions in neurobiology. The principal attributes which have made the HEK cell a popular choice among electrophysiologists to study isolated receptor channels include; its quick and easy reproduction and maintenance; amenability to transfection using a wide variety of methods; high efficiency of transfection and protein production; faithful translation and processing of proteins; and small cell size with minimal processes appropriate for voltage-clamp experimentation. These, and other attributes, also mean that complementary biochemical/cell biological evaluations of expressed proteins can be performed in concert with functional analyses to establish detailed pharmacological and biophysical profiles for the action of new drugs and their targets. The increased amount of sequence information available from the human genome has placed greater emphasis upon heterologous cell expression systems as targets for high throughput structure-function evaluation of novel drug targets and disease markers. Here we have highlighted some of the innate characteristics of the HEK cell in order that its suitability as a vehicle for the expression of a gene product can be assessed for particular needs. We have also detailed some of the standard methods used for transfection and obtaining functional data from electrophysiological recording techniques.

  14. Absence of keratins 8 and 18 expression in rodent epithelial cell lines associates with keratin gene mutation and DNA methylation: cell line selective effects on cell invasion

    PubMed Central

    Omary, M. Bishr

    2016-01-01

    Epithelial-mesenchymal transition (EMT) in carcinoma is associated with dramatic up-regulation of vimentin and down-regulation of the simple-type keratins 8 and 18 (K8/K18), but the mechanisms of these changes are poorly understood. We demonstrate that two commonly-studied murine (CT26) and rat (IEC-6) intestinal cell lines have negligible K8/K18 but high vimentin protein expression. Proteasome inhibition led to a limited increase in K18 but not K8 stabilization, thereby indicating that K8/K18 absence is not due, in large part, to increased protein turnover. CT26 and IEC-6 cells had <10% of normal K8/K18 mRNA and exhibited decreased mRNA stability, with K8 being higher in IEC-6 versus CT26 and K18 being higher in CT26 versus IEC-6 cells. Keratin gene sequencing showed that KRT8 in CT26 cells had a 21-nucleotide deletion while K18 in IEC-6 cells had a 9-amino acid in-frame insertion. Furthermore, the KRT8 promoter in CT26 and the KRT18 promoter in IEC-6 are hypermethylated. Inhibition of DNA methylation using 5-azacytidine increased K8 or K18 in some but all the tested rodent epithelial cell lines. Restoring K8 and K18 by lentiviral transduction reduced CT26 but not IEC-6 cell matrigel invasion. K8/K18 re-introduction also decreased E-cadherin expression in IEC-6 but not CT26 cells, suggesting that the effect of keratin expression on epithelial to mesenchymal transition is cell-line dependent. Therefore, some commonly utilized rodent epithelial cell lines, unexpectedly, manifest barely detectable keratin expression but have high levels of vimentin. In the CT26 and IEC-6 intestinal cell lines, keratin expression correlates with keratin gene insertion or deletion and with promoter methylation, which likely suppress keratin transcription or mRNA stability. PMID:25882495

  15. LINE-1 retrotransposition events regulate gene expression after X-ray irradiation.

    PubMed

    Banaz-Yaşar, Ferya; Gedik, Nilgün; Karahan, Selda; Diaz-Carballo, David; Bongartz, Birthe M; Ergün, Süleyman

    2012-09-01

    Long interspersed nuclear element-1 (LINE-1) retrotransposons are mobile elements that insert into new genomic locations via reverse transcription of an RNA intermediate. The mechanism of retrotransposition is not entirely understood. The integration of these elements occurs by target-primed reverse transcription (TPRT), which initiates double-strand breaks (DSBs) during the LINE-1 integration. Also, X-ray is known to induce DNA damage. The aim of this study was to evaluate the potential effects of LINE-1 de novo retrotransposition on the expression of different genes after X-ray irradiation in human endothelial cells. After stable transfection of the human hybrid endothelial cell line EA.hy926 with the human LINE-1 element, we analyzed the expression of different genes after irradiation with 5 Gy X-rays by reverse transcription-polymerase chain reaction (RT-PCR). We determine the expression level of phosphorylated p53 and γ-histone H2AX protein levels upon X-ray irradiation with 5 Gy for 24 h. Our results showed that EA.hy926 LINE-1 cell clones react with a strong upregulation of phosphorylated p53 protein, already 15 min after irradiation compared to the wild type (WT) cells. Also, the expression of γ-histone H2AX protein was elevated in the cell clones with retrotransposition events 15 min after irradiation, whereas the WT cells have a delayed expression of phosphorylated histone H2AX protein. Taken together, our findings provide that LINE-1 retrotransposition events regulate different gene expression after irradiation in the EA.hy926 cell line.

  16. Comparative Membranome expression analysis in primary tumors and derived cell lines.

    PubMed

    Uva, Paolo; Lahm, Armin; Sbardellati, Andrea; Grigoriadis, Anita; Tutt, Andrew; de Rinaldis, Emanuele

    2010-01-01

    Despite the wide use of cell lines in cancer research, the extent to which their surface properties correspond to those of primary tumors is poorly characterized. The present study addresses this problem from a transcriptional standpoint, analyzing the expression of membrane protein genes--the Membranome--in primary tumors and immortalized in-vitro cultured tumor cells. 409 human samples, deriving from ten independent studies, were analyzed. These comprise normal tissues, primary tumors and tumor derived cell lines deriving from eight different tissues: brain, breast, colon, kidney, leukemia, lung, melanoma, and ovary. We demonstrated that the Membranome has greater power than the remainder of the transcriptome when used as input for the automatic classification of tumor samples. This feature is maintained in tumor derived cell lines. In most cases primary tumors show maximal similarity in Membranome expression with cell lines of same tissue origin. Differences in Membranome expression between tumors and cell lines were analyzed also at the pathway level and biological themes were identified that were differentially regulated in the two settings. Moreover, by including normal samples in the analysis, we quantified the degree to which cell lines retain the Membranome up- and down-regulations observed in primary tumors with respect to their normal counterparts. We showed that most of the Membranome up-regulations observed in primary tumors are lost in the in-vitro cultured cells. Conversely, the majority of Membranome genes down-regulated upon tumor transformation maintain lower expression levels also in the cell lines. This study points towards a central role of Membranome genes in the definition of the tumor phenotype. The comparative analysis of primary tumors and cell lines identifies the limits of cell lines as a model for the study of cancer-related processes mediated by the cell surface. Results presented allow for a more rational use of the cell lines as a

  17. Lipopolysaccharide induces multinuclear cell from RAW264.7 line with increased phagocytosis activity

    SciTech Connect

    Nakanishi-Matsui, Mayumi; Yano, Shio; Matsumoto, Naomi; Futai, Masamitsu

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer LPS induces multinuclear cells from murine macrophage-derived RAW264.7 cells. Black-Right-Pointing-Pointer The multinuclear cells are formed through cell-cell fusion in the presence of Ca{sup 2+}. Black-Right-Pointing-Pointer The multinuclear cells do not express osteoclast-specific enzymes. Black-Right-Pointing-Pointer They internalized more and larger beads than mononuclear cells and osteoclasts. -- Abstract: Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, induces strong proinflammatory responses, including the release of cytokines and nitric oxide from macrophage. In this study, we found that a murine macrophage-derived line, RAW264.7, became multinuclear through cell-cell fusion after incubation with highly purified LPS or synthetic lipid A in the presence of Ca{sup 2+}. The same cell line is known to differentiate into multinuclear osteoclast, which expresses a specific proton pumping ATPase together with osteoclast markers on stimulation by the extracellular domain of receptor activator of nuclear factor {kappa}B ligand (Toyomura, T., Murata, Y., Yamamoto, A., Oka, T., Sun-Wada, G.-H., Wada, Y. and Futai, M., 2003). The LPS-induced multinuclear cells did not express osteoclast-specific enzymes including tartrate-resistant acid phosphatase and cathepsin K. During multinuclear cell formation, the cells internalized more and larger polystyrene beads (diameter 6-15 {mu}m) than mononuclear cells and osteoclasts. The internalized beads were located in lysosome-marker positive organelles, which were probably phagolysosomes. The LPS-induced multinuclear cell could be a good model system to study phagocytosis of large foreign bodies.

  18. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    SciTech Connect

    Medina, D.; Oborn, C.J. ); Li, M.L.; Bissell, M.J. )

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  19. Expression Profile of MiR-128 in the Astrocytoma Patients and Cell Lines.

    PubMed

    Xu, Jingjing; Liu, Yuqiong; Guo, Si; Ma, Shengli; Xiao, Lin; Wei, Na; Xue, Rui

    2016-09-01

    Malignant astrocytomas are the most common primary brain tumors. The critical characterizes of astrocyomas are their aggressive and infiltrative in the brain, which leads to uncontrollable by conventional forms of therapy. MicroRNAs are small RNAs that had been found to regulate their targets by specific binding to the 3'-untranslated region (3'UTR) of mRNA. Recent advances in understanding the molecular biology of these tumors have revealed that microRNA (miRNA) disruption may play important roles in the pathogenesis of astrocytomas. And some of the miRNA alterations were found in the serum of astrocytoma patients. In this study, we studied the expression profile of miR-128, in the different stages of astrocytoma tissues and two human astrocytoma cell lines, A172 and T98G cells. We found that the levels of miR-128 are decreased in the A172 and T98G cells when compared to normal human astrocyte (NHA). Furthermore, the levels of miR-128 decreased gradually to the pathological stages of astrocytomas. We also identified that TROVE2 is a novel target of miR-128 by the luciferase reporter system. Furthermore, the expression levels of TROVE2 are dramatically increased with the pathological stages increasing. Finally, the levels of TROVE2 are negatively correlated with miR-128 in astrocytoma tissues. Our data provided novel evidence for the miR-128 and TROVE2 in the development of human astrocytomas. PMID:26307612

  20. Spatiotemporal calcium signaling in a Drosophila melanogaster cell line stably expressing a Drosophila muscarinic acetylcholine receptor.

    PubMed

    Cordova, D; Delpech, V Raymond; Sattelle, D B; Rauh, J J

    2003-11-01

    A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca(2+)](i)) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca(2+)](i) transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx. PMID:12827518

  1. Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression

    PubMed Central

    Suchanski, Jaroslaw; Olbromski, Mateusz; Gomulkiewicz, Agnieszka; Owczarek, Tomasz; Kruczak, Anna; Ambicka, Aleksandra; Rys, Janusz; Ugorski, Maciej; Podhorska-Okolow, Marzena; Dziegiel, Piotr

    2015-01-01

    It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients’ shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases. PMID:25933064

  2. A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene.

    PubMed Central

    Sutherland, H G; Martin, D I; Whitelaw, E

    1997-01-01

    Enhancer elements have been shown to affect the probability of a gene establishing an active transcriptional state and suppress the silencing of reporter genes in cell lines, but their effect in transgenic mice has been obscured by the use of assays that do not assess expression on a cell-by-cell basis. We have examined the effect of a globin enhancer on the variegation of lacZ expression in erythrocytes of transgenic mice. Mice carrying lacZ driven by the alpha-globin promoter exhibit beta-galactosidase (beta-Gal) expression in only a very small proportion of embryonic erythrocytes. When the transgenic construct also contains the (alphaHS-40 enhancer, which controls expression of the alpha-globin gene, expression is seen in a high proportion of embryonic erythrocytes, although there are variations between transgenic lines which can be attributed to different sites of integration. Analysis of beta-Gal expression levels suggests that expressing cells in lines carrying only the alpha-globin promoter express as much beta-Gal as those in which the transgene also contains alphaHS-40. A marked decline in transgene expression occurs as mice age, which is mainly due to a decrease in the proportion of cells expressing the transgene. Thus, a globin enhancer can act to suppress variegation of a linked transgene; this result is consistent with a model in which enhancers act to establish and maintain an active domain without directly affecting the transcriptional rate. PMID:9032288

  3. Haloperidol, but not olanzapine, may affect expression of PER1 and CRY1 genes in human glioblastoma cell line

    PubMed Central

    Mokros, Łukasz; Karbownik, Michał Seweryn; Nowakowska-Domagała, Katarzyna; Szemraj, Janusz; Wieteska, Łukasz; Woźniak, Karol; Witusik, Andrzej; Antczak, Adam; Pietras, Tadeusz

    2016-01-01

    Abstract Background: There is barely any evidence of antipsychotic drugs affecting the molecular clockwork in human, yet it is suggested that clock genes are associated with dopaminergic transmission, i.e. the main target of this therapeutics. We decided to verify if haloperidol and olanzapine affect expression of CLOCK, BMAL1, PER1 and CRY1 in a human central nervous system cell line model. Methods: U-87MG human glioblastoma cell line was used as an experimental model. The cells were incubated with or without haloperidol and olanzapine in the concentration of 5 and 20 μM for 24 h. Real-time quantitative polymerase chain reaction with the ΔC T analysis was used to examine the effect of haloperidol and olanzapine on the mRNA expression of the genes. Results: At 5 μM, haloperidol decreased expression of CRY1 almost 20-fold. There was nearly a 1.5-fold increase in expression of PER1. Considering the 20 μM haloperidol concentration and both olanzapine concentrations, no other statistically significant effect was observed. Conclusions: At certain concentration, haloperidol seems to affect expression of particular clock genes in a human central nervous system cell line model, yet mechanism underlying this phenomenon remains elusive.

  4. Expression profile of hypothalamic neuropeptides in chicken lines selected for high or low residual feed intake.

    PubMed

    Sintubin, P; Greene, E; Collin, A; Bordas, A; Zerjal, T; Tesseraud, S; Buyse, J; Dridi, S

    2014-08-01

    The R(+) and R(-) chicken lines have been divergently selected for high (R(+)) or low (R(-)) residual feed intake. For the same body weight and egg production, the R(+) chickens consume 40% more food than their counterparts R(-) lines. In the present study we sought to determine the hypothalamic expression profile of feeding-related neuropeptides in these lines maintained under fed or food-deprived conditions. In the fed condition, the suppressor of cytokine signaling 3 (SOCS3) was 17-fold lower (P<0.05) and the ghrelin receptor was 7-fold higher (P<0.05) in R(+) compared to R(-) chicken lines. The hypothalamic expression of the other studied genes remained unchanged between the two lines. In the fasted state, orexigenic neuropeptide Y and agouti-related peptide were more responsive, with higher significant levels in the R(+) compared to R(-) chickens, while no significant differences were seen for the anorexigenic neuropeptides pro-opiomelanocortin and corticotropin releasing hormone. Interestingly, C-reactive protein, adiponectin receptor 1 and ghrelin receptor gene expression were significantly higher (12-, 2- and 3-folds, respectively), however ghrelin and melanocortin 5 receptor mRNA levels were lower (4- and 2-folds, P=0.05 and P=0.03, respectively) in R(+) compared to R(-) animals. We identified several key feeding-related genes that are differently expressed in the hypothalamus of R(+) and R(-) chickens and that might explain the difference in feed intake observed between the two lines.

  5. Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance

    PubMed Central

    2012-01-01

    Background Cancer-initiating cells (CICs) are proposed to be responsible for the generation of metastasis and resistance to therapy. Accumulating evidences indicates CICs are found among different human cancers and cell lines derived from them. Few studies address the characteristics of CICs in cervical cancer. We identify biological features of CICs from four of the best-know human cell lines from uterine cervix tumors. (HeLa, SiHa, Ca Ski, C-4 I). Methods Cells were cultured as spheres under stem-cell conditions. Flow cytometry was used to detect expression of CD34, CD49f and CD133 antigens and Hoechst 33342 staining to identify side population (SP). Magnetic and fluorescence-activated cell sorting was applied to enrich and purify populations used to evaluate tumorigenicity in nude mice. cDNA microarray analysis and in vitro radioresistance assay were carried out under standard conditions. Results CICs, enriched as spheroids, were capable to generate reproducible tumor phenotypes in nu-nu mice and serial propagation. Injection of 1 × 103 dissociated spheroid cells induced tumors in the majority of animals, whereas injection of 1 × 105 monolayer cells remained nontumorigenic. Sphere-derived CICs expressed CD49f surface marker. Gene profiling analysis of HeLa and SiHa spheroid cells showed up-regulation of CICs markers characteristic of the female reproductive system. Importantly, epithelial to mesenchymal (EMT) transition-associated markers were found highly expressed in spheroid cells. More importantly, gene expression analysis indicated that genes required for radioresistance were also up-regulated, including components of the double-strand break (DSB) DNA repair machinery and the metabolism of reactive oxygen species (ROS). Dose-dependent radiation assay indicated indeed that CICs-enriched populations exhibit an increased resistance to ionizing radiation (IR). Conclusions We characterized a self-renewing subpopulation of CICs found among four well known human

  6. Characterization of kinase suppressor of Ras-1 expression and anticancer drug sensitivity in human cancer cell lines.

    PubMed

    Stoeger, Scott M; Cowan, Kenneth H

    2009-04-01

    Previous studies have indicated that the ERK1/2 MAP kinase signaling pathway plays an important role not only in cell growth, cell cycle regulation, and differentiation, but also in determining the sensitivity of cells to anticancer agents as well. Furthermore, expression of kinase suppressor of Ras-1 (KSR1), a molecular scaffold that modulates signaling through the ERK1/2 MAP kinase pathway, has been shown to influence the cellular sensitivity to the anticancer agent cisplatin. To further define the role of KSR1 expression on drug sensitivity, the expression of KSR1 was examined in the NCI60 anticancer drug screen, a panel of cancer cell lines representing nine tissue types, established by the Developmental Therapeutics Program (DTP) at the National Cancer Institute (NCI). The expression of thousands of molecular targets has been examined in the NCI60 panel as well as the cellular toxicity for greater than 400,000 compounds. KSR1 expression varied almost 30-fold difference between the highest and lowest expressing cell lines in the NCI60. Using the COMPARE analysis algorithm, KSR1 expression was correlated with sensitivity of the compounds screened by DTP and several novel agents were identified whose sensitivity correlated with KSR1 expression in the NCI60 panel. Cytotoxicity of two agents, cytochalasin H and tunicamycin, identified through the COMPARE analysis of KSR1 expression and drug sensitivity, was also examined in wild type (KSR(+/+)) mouse embryo fibroblasts (MEFs) and MEFs deficient in KSR1 expression (KSR1(-/-)). These studies demonstrated enhanced sensitivity, as well as increased ERK activation, in KSR(-/-) MEFs following exposure to tunicamycin or cytochalasin H compared to KSR(+/+) MEFs. Furthermore, restoration of KSR1 expression in KSR(-/-) MEFs following stable transduction of cells with a KSR1 expression vector, enhanced sensitivity of cells to tunicamycin and cytochalasin H and decreased ERK1/2 activation following exposure to these drugs. In

  7. Morphine-Induced Constipation Develops With Increased Aquaporin-3 Expression in the Colon via Increased Serotonin Secretion.

    PubMed

    Kon, Risako; Ikarashi, Nobutomo; Hayakawa, Akio; Haga, Yusuke; Fueki, Aika; Kusunoki, Yoshiki; Tajima, Masataka; Ochiai, Wataru; Machida, Yoshiaki; Sugiyama, Kiyoshi

    2015-06-01

    Aquaporin-3 (AQP3) is a water channel that is predominantly expressed in the colon, where it plays a critical role in the regulation of fecal water content. This study investigated the role of AQP3 in the colon in morphine-induced constipation. AQP3 expression levels in the colon were analyzed after oral morphine administration to rats. The degree of constipation was analyzed after the combined administration of HgCl(2) (AQP3 inhibitor) or fluoxetine (5-HT reuptake transporter [SERT] inhibitor) and morphine. The mechanism by which morphine increased AQP3 expression was examined in HT-29 cells. AQP3 expression levels in rat colon were increased during morphine-induced constipation. The combination of HgCl(2) and morphine improved morphine-induced constipation. Treatment with morphine in HT-29 cells did not change AQP3 expression. However, 5-HT treatment significantly increased the AQP3 expression level and the nuclear translocation of peroxisome proliferator-activated receptor gamma (PPARγ) 1 h after treatment. Pretreatment with fluoxetine significantly suppressed these increases. Fluoxetine pretreatment suppressed the development of morphine-induced constipation and the associated increase in AQP3 expression in the colon. The results suggest that morphine increases the AQP3 expression level in the colon, which promotes water absorption from the luminal side to the vascular side and causes constipation. This study also showed that morphine-induced 5-HT secreted from the colon was taken into cells by SERT and activated PPARγ, which subsequently increased AQP3 expression levels.

  8. Comparative study of SOS2 and a novel PMP3-1 gene expression in two sunflower (Helianthus annuus L.) lines differing in salt tolerance.

    PubMed

    Saadia, Mubshara; Jamil, Amer; Ashraf, Muhammad; Akram, Nudrat Aisha

    2013-06-01

    Gene expression pattern of two important regulatory proteins, salt overly sensitive 2 (SOS2) and plasma membrane protein 3-1 (PMP3-1), involved in ion homeostasis, was analyzed in two salinity-contrasting sunflower (Helianthus annuus L.) lines, Hysun-38 (salt tolerant) and S-278 (moderately salt tolerant). The pattern was studied at selected time intervals (24 h) under 150 mM NaCl treatment. Using reverse transcription PCR, SOS2 gene fragment was obtained from young leaf and root tissues of opposing lines while that for PMP3-1 was obtained only from young root tissues. Both tolerant and moderately tolerant lines showed a gradual increase in SOS2 expression in sunflower root tissues. Leaf tissues showed the gradually increasing pattern of SOS2 expression in tolerant plants as compared to that for moderately tolerant ones that showed a relatively lower level of expression for this gene. We found the highest level of PMP 3-1 expression in the roots of tolerant sunflower line at 6 and 12 h postsalinity treatment. The moderately tolerant line showed higher expression of PMP3-1 at 12 and 24 h after salt treatment. Overall, the expression of genes for both the regulator proteins varied significantly in the two sunflower lines differing in salinity tolerance.

  9. Increasing morphinan alkaloid production by over-expressing codeinone reductase in transgenic Papaver somniferum.

    PubMed

    Larkin, Philip J; Miller, James A C; Allen, Robert S; Chitty, Julie A; Gerlach, Wayne L; Frick, Susanne; Kutchan, Toni M; Fist, Anthony J

    2007-01-01

    Only plants of the Papaver genus (poppies) are able to synthesize morphinan alkaloids, and cultivation of P. somniferum, opium poppy, remains critical for the production and supply of morphine, codeine and various semi-synthetic analgesics. Opium poppy was transformed with constitutively expressed cDNA of codeinone reductase (PsCor1.1), the penultimate step in morphine synthesis. Most transgenic lines showed significant increases in capsule alkaloid content in replicated glasshouse and field trials over 4 years. The morphinan alkaloid contents on a dry weight basis were between 15% and 30% greater than those in control high-yielding genotypes and control non-transgenic segregants. Transgenic leaves had approximately 10-fold greater levels of Cor transcript compared with non-transgenic controls. Two cycles of crossing of the best transgenic line into an elite high-morphine genotype resulted in significant increases in morphine and total alkaloids relative to the elite recurrent parent. No significant changes in alkaloid profiles or quantities were observed in leaf, roots, pollen and seed.

  10. A nanoemulsion formulation of tamoxifen increases its efficacy in a breast cancer cell line.

    PubMed

    Tagne, Jean-Bosco; Kakumanu, Srikanth; Ortiz, Daniela; Shea, Thomas; Nicolosi, Robert J

    2008-01-01

    This paper reports on the preparation of a water-soluble nanoemulsion of the highly lipid-soluble drug tamoxifen (TAM). In addition, relative to a suspension of TAM, the nanoemulsion preparation demonstrated a greater zeta potential (increased negative charge) which has previously been associated with increasing drug/membrane permeability. This study also reports that relative to suspensions of TAM with particle sizes greater than 6000 nm, nanoemulsions of TAM, having mean particle sizes of 47 nm, inhibited cell proliferation 20-fold greater and increased cell apoptosis 4-fold greater in the HTB-20 breast cancer cell line. Thus, this work suggests that a nanoemulsion compared to a suspension preparation of TAM increases its anticancer properties relative to breast cancer. PMID:18171014

  11. c-Myc over-expression in Ramos Burkitt's lymphoma cell line predisposes to iron homeostasis disruption in vitro

    SciTech Connect

    Habel, Marie-Eve; Jung, Daniel . E-mail: djung@hema-quebec.qc.ca

    2006-03-24

    Burkitt's lymphoma is an aggressive B-cell neoplasm resulting from deregulated c-myc expression. We have previously shown that proliferation of Burkitt's lymphoma cell lines such as Ramos is markedly reduced by iron treatment. It has been shown that iron induces expression of c-myc which, owing to its transcriptional regulatory functions, regulates genes involved in iron metabolism. Transient enhancement of c-myc expression by iron could increase the expression of genes involved in iron incorporation, which could lead to an accumulation of intracellular free iron. Here, we have investigated whether cells with a high basal level of c-Myc were more likely to accumulate free iron. Our results suggest that the basal level of c-Myc in Ramos cells is twofold higher than what is seen in HL-60 cells. Moreover, in Ramos cells, where c-Myc is expressed at a high level, H-ferritin expression is down-regulated, transferrin receptor (CD71) expression is increased, and ferritin translation is inhibited. These modifications in iron metabolism, resulting from the strong basal expression of c-Myc, and amplified by iron addition, could lead to a disruption in homeostasis and consequently to growth arrest.

  12. Cerium oxide nanoparticles inhibit the migration and proliferation of gastric cancer by increasing DHX15 expression

    PubMed Central

    Xiao, Yu-Feng; Li, Jian-Mei; Wang, Su-Min; Yong, Xin; Tang, Bo; Jie, Meng-Meng; Dong, Hui; Yang, Xiao-Chao; Yang, Shi-Ming

    2016-01-01

    Gastric cancer is one of the leading causes of tumor-related deaths in the world. Current treatment options do not satisfy doctors and patients, and new therapies are therefore needed. Cerium oxide nanoparticles (CNPs) have been studied as a potential therapeutic approach for treating many diseases. However, their effects on human gastric cancer are currently unknown. Therefore, in this study, we aimed to characterize the effects of CNPs on human gastric cancer cell lines (MKN28 and BGC823). Gastric cancer cells were cocultured with different concentrations of CNPs, and proliferation and migration were measured both in vitro and in vivo. We found that CNPs inhibited the migration of gastric cancer cells when applied at different concentrations, but only a relatively high concentration (10 µg/mL) of CNPs suppressed proliferation. Furthermore, we found that CNPs increased the expression of DHX15 and its downstream signaling pathways. We therefore provide evidence showing that CNPs may be a promising approach to suppress malignant activity of gastric cancer by increasing the expression of DHX15. PMID:27486320

  13. [Comparative study of therapy targeted genes expression in neuroblastoma cell lines].

    PubMed

    Lebedev, T D; Spirin, P V; Orlova, N N; Prokofjeva, M M; Prassolov, V S

    2015-01-01

    In this study we evaluated c-kit, VEGFA, and MYC gene expression level in seven neuroblastoma stable cell lines: SK-N-SH, SK-N-BE, SK-N-AS, SH-SY5Y, Kelly, IMR-32, and LAN-1. Expression levels of these genes can serve as diagnostic factors of cancer progression, and proteins encoded by these genes are promising targets for neuroblastoma treatment. SH-SY5Y and SK-N-AS cells have highest MYC expression and the same VEGFA expression, although SH-SY5Y has 10 times higher c-kit expression than SK-N-AS cells. Both IMR-32 and LAN-1 cells have low MYC expression level, but differ in c-kit expression, IMR-32 has significantly higher c-kit expression, than any other neuroblastoma cell line. LAN-1 on the other hand has the highest VEGFA expression. These data suggest that MYC, c-kit, and VEGFA genes can play different roles in development and progression of neuroblastoma depending on other activated molecular mechanisms in malignant cells.

  14. PTEN expression in PTEN-null leukaemic T cell lines leads to reduced proliferation via slowed cell cycle progression.

    PubMed

    Seminario, Maria-Cristina; Precht, Patricia; Wersto, Robert P; Gorospe, Myriam; Wange, Ronald L

    2003-11-01

    The balance of activities between the proto-oncogene phosphoinositide 3-kinase (PI3K) and the tumour suppressor gene PTEN has been shown to affect cellular growth and proliferation, as well as tumorigenesis. Previously, PTEN expression in the PTEN-null Jurkat T cell leukaemia line was shown to cause reduced proliferation without cell cycle arrest. Here, we further these investigations by determining the basis for this phenomenon. By BrdU pulse-chase and cell cycle arrest and release assays, we find that PTEN expression reduced proliferation by slowing progression through all phases of the cell cycle. This was associated with reduced levels of cyclins A, B1 and B2, cdk4, and cdc25A and increased p27KIP1 expression. Apoptosis played no role in the antiproliferative effect of PTEN, since only marginal increases in the rate of apoptosis were detected upon PTEN expression, and inhibitors of effector caspases did not restore proliferative capacity. Active Akt blocked the antiproliferative effects of PTEN, indicating that PTEN mediates its effects through conventional PI3K-linked signalling pathways. Similar results were obtained from a different PTEN-null leukaemia T cell line, CEM. Together, these results show that PTEN expression in leukaemic T cells leads to reduced proliferation via an apoptosis-independent mechanism involving slower passage through the cell cycle.

  15. Zidovudine, abacavir and lamivudine increase the radiosensitivity of human esophageal squamous cancer cell lines.

    PubMed

    Chen, Xuan; Wang, Cong; Guan, Shanghui; Liu, Yuan; Han, Lihui; Cheng, Yufeng

    2016-07-01

    Telomerase is a type of reverse transcriptase that is overexpressed in almost all human tumor cells, but not in normal tissues, which provides an opportunity for radiosensitization targeting telomerase. Zidovudine, abacavir and lamivudine are reverse transcriptase inhibitors that have been applied in clinical practice for several years. We sought to explore the radiosensitization effect of these three drugs on human esophageal cancer cell lines. Eca109 and Eca9706 cells were treated with zidovudine, abacavir and lamivudine for 48 h before irradiation was administered. Samples were collected 1 h after irradiation. Clonal efficiency assay was used to evaluate the effect of the combination of these drugs with radiation doses of 2, 4, 6 and 8 Gy. DNA damage was measured by comet assay. Telomerase activity (TA) and relative telomere length (TL) were detected and evaluated by real-time PCR. Apoptosis rates were assessed by flow cytometric analysis. The results showed that all the drugs tested sensitized the esophageal squamous cell carcinoma (ESCC) cell lines to radiation through an increase in radiation-induced DNA damage and cell apoptosis, deregulation of TA and decreasing the shortened TL caused by radiation. Each of the drugs investigated (zidovudine, abacavir and lamivudine) could be used for sensitizing human esophageal cancer cell lines to radiation. Consequently, the present study supports the potential of these three drugs as therapeutic agents for the radiosensitization of esophageal squamous cell cancer. PMID:27220342

  16. Activation of the Farnesoid X-receptor in breast cancer cell lines results in cytotoxicity but not increased migration potential.

    PubMed

    Alasmael, Noura; Mohan, Rati; Meira, Lisiane B; Swales, Karen E; Plant, Nick J

    2016-01-28

    Breast cancer is the commonest form of cancer in women, but successful treatment is confounded by the heterogeneous nature of breast tumours: Effective treatments exist for hormone-sensitive tumours, but triple-negative breast cancer results in poor survival. An area of increasing interest is metabolic reprogramming, whereby drug-induced alterations in the metabolic landscape of a tumour slow tumour growth and/or increase sensitivity to existing therapeutics. Nuclear receptors are transcription factors central to the expression of metabolic and transport proteins, and thus represent potential targets for metabolic reprogramming. We show that activation of the nuclear receptor FXR, either by its endogenous ligand CDCA or the synthetic GW4064, leads to cell death in four breast cancer cell lines with distinct phenotypes: MCF-10A (normal), MCF-7 (receptor positive), MDA-MB-231 and MDA-MB-468 (triple negative). Furthermore, we show that the mechanism of cell death is predominantly through the intrinsic apoptotic pathway. Finally, we demonstrate that FXR agonists do not stimulate migration in breast cancer cell lines, an important potential adverse effect. Together, our data support the continued examination of FXR agonists as a novel class of therapeutics for the treatment of breast cancer.

  17. Effect of oxygen on the expression of renin-angiotensin system components in a human trophoblast cell line.

    PubMed

    Delforce, Sarah J; Wang, Yu; Van-Aalst, Meg E; Corbisier de Meaultsart, Celine; Morris, Brian J; Broughton-Pipkin, Fiona; Roberts, Claire T; Lumbers, Eugenie R; Pringle, Kirsty G

    2016-01-01

    During the first trimester, normal placental development occurs in a low oxygen environment that is known to stimulate angiogenesis via upregulation of vascular endothelial growth factor (VEGF). Expression of the placental renin-angiotensin system (RAS) is highest in early pregnancy. While the RAS and oxygen both stimulate angiogenesis, how they interact within the placenta is unknown. We postulated that low oxygen increases expression of the proangiogenic RAS pathway and that this is associated with increased VEGF in a first trimester human trophoblast cell line (HTR-8/SVneo). HTR-8/SVneo cells were cultured in one of three oxygen tensions (1%, 5% and 20%). RAS and VEGF mRNA expression were determined by qPCR. Prorenin, angiotensin converting enzyme (ACE) and VEGF protein levels in the supernatant, as well as prorenin and ACE in cell lysates, were measured using ELISAs. Low oxygen significantly increased the expression of both angiotensin II type 1 receptor (AGTR1) and VEGF (both P < 0.05). There was a positive correlation between AGTR1 and VEGF expression at low oxygen (r = 0.64, P < 0.005). Corresponding increases in VEGF protein were observed with low oxygen (P < 0.05). Despite no change in ACE1 mRNA expression, ACE levels in the supernatant increased with low oxygen (1% and 5%, P < 0.05). Expression of other RAS components did not change. Low oxygen increased AGTR1 and VEGF expression, as well as ACE and VEGF protein levels, suggesting that the proangiogenic RAS pathway is activated. This highlights a potential role for the placental RAS in mediating the proangiogenic effects of low oxygen in placental development.

  18. Increased Expression of Bcl11b Leads to Chemoresistance Accompanied by G1 Accumulation

    PubMed Central

    Grabarczyk, Piotr; Nähse, Viola; Delin, Martin; Przybylski, Grzegorz; Depke, Maren; Hildebrandt, Petra; Völker, Uwe; Schmidt, Christian A.

    2010-01-01

    Background The expression of BCL11B was reported in T-cells, neurons and keratinocytes. Aberrations of BCL11B locus leading to abnormal gene transcription were identified in human hematological disorders and corresponding animal models. Recently, the elevated levels of Bcl11b protein have been described in a subset of squameous cell carcinoma cases. Despite the rapidly accumulating knowledge concerning Bcl11b biology, the contribution of this protein to normal or transformed cell homeostasis remains open. Methodology/Principal Findings Here, by employing an overexpression strategy we revealed formerly unidentified features of Bcl11b. Two different T-cell lines were forced to express BCL11B at levels similar to those observed in primary T-cell leukemias. This resulted in markedly increased resistance to radiomimetic drugs while no influence on death-receptor apoptotic pathway was observed. Apoptosis resistance triggered by BCL11B overexpression was accompanied by a cell cycle delay caused by accumulation of cells at G1. This cell cycle restriction was associated with upregulation of CDKN1C (p57) and CDKN2C (p18) cyclin dependent kinase inhibitors. Moreover, p27 and p130 proteins accumulated and the SKP2 gene encoding a protein of the ubiquitin-binding complex responsible for their degradation was repressed. Furthermore, the expression of the MYCN oncogene was silenced which resulted in significant depletion of the protein in cells expressing high BCL11B levels. Both cell cycle restriction and resistance to DNA-damage-induced apoptosis coincided and required the histone deacetylase binding N-terminal domain of Bcl11b. The sensitivity to genotoxic stress could be restored by the histone deacetylase inhibitor trichostatine A. Conclusions The data presented here suggest a potential role of BCL11B in tumor survival and encourage developing Bcl11b-inhibitory approaches as a potential tool to specifically target chemoresistant tumor cells. PMID:20824091

  19. Modulation of homeobox B6 and B9 genes expression in human leukemia cell lines during myelomonocytic differentiation.

    PubMed

    Ohnishi, K; Tobita, T; Sinjo, K; Takeshita, A; Ohno, R

    1998-11-01

    Homeobox genes (HOX) may have a regulatory function in the differentiation process of hematopoiesis. We examined the change of HOX B6 and HOX B9 mRNA expressions during the in vitro differentiation of four myeloid leukemia cell lines because HOX B6 may be involved closely in myeloid differentiation. HL-60, NB4, NKM-1 and NOMO-1 were established from acute leukemia of M2, M3, M2 and M5 subtype of the French-American-British classification, respectively. All-trans retinoic acid (ATRA), TPA, and G-CSF were used as differentiation inducers. Each cell line was cultured with each inducer and total RNA was isolated on day 1, 2, 3, or 5. HOX B mRNA was detected by Northern blotting and RT-PCR methods. HOX B6 and HOX B9 mRNAs were constitutively expressed in NB4, NKM-1 and NOMO-1, but were expressed at very low levels in HL-60. HOX B6 and HOX B9 mRNAs were also expressed in fresh acute myelocytic leukemia blasts. HOX B6 mRNA expression in HL-60, NB4, and NKM-1 cultured with ATRA increased on day 3 and decreased on day 5. HOX B6 mRNA expression in NB4 and NKM-1 cultured with TPA decreased on day 3. HOX B9 mRNA expression displayed changes similar to those of HOX B6 mRNA in NB4 and NKM-1. These results indicate that myeloid leukemia cell lines express HOX B6 and HOX B9, and that their respective mRNA expressions in NB4 and HL-60 increase at a mid stage of myeloid differentiation by ATRA induction and then decrease during a late stage. HOX B6 mRNA expression decreased in monocytoid differentiation by TPA induction in NB4, HL-60 and NKM-1. HOX B6 antisense-oligonucleotide inhibited the proliferation of NB4 and NKM-1. These results suggest that HOX B gene expression is related to simultaneous activation of cellular proliferation and differentiation in leukemic cells. PMID:9922051

  20. Suppression of laminin-5 expression leads to increased motility, tumorigenicity, and invasion

    SciTech Connect

    Yuen Hengwai; Ziober, Amy F.; Gopal, Pallavi; Nasrallah, Ilya; Falls, Erica M.; Meneguzzi, Guerrino; Ang, Hwee-Quan; Ziober, Barry L. . E-mail: bziober@mail.med.upenn.edu

    2005-09-10

    Laminin-5 (Ln-5) is expressed in several human carcinomas and hypothesized to contribute to tumor invasion. To understand the role of Ln-5 in human cancers, we stably delivered small interfering RNAs (siRNAs) directed against the Ln-5 {gamma}2 chain into JHU-022-SCC cells (022), a non-invasive oral squamous cell carcinoma (OSCC) cell line which secretes Ln-5. Lysates from {gamma}2 siRNA cells (022-si{gamma}2) had nearly undetectable levels of the {gamma}2 chain while the {alpha}3 and {beta}3 subunits of Ln-5 remained unchanged compared to parental and control. In conditioned medium from 022-si{gamma}2 cells, the {gamma}2 chain and the Ln-5 heterotrimer were barely detectable, similar to an invasive OSCC cell line. Conditioned medium from 022-si{gamma}2 cells contained less {alpha}3 and {beta}3 subunits than both parental and control. Although the proliferation and adhesive properties of the 022-si{gamma}2 cells remained similar to parental and control cells, 022-si{gamma}2 cells showed increased detachment and a fibroblastic morphology similar to invasive cells. Moreover, migration, in vitro invasion, and in vivo tumorigenicity were enhanced in 022-si{gamma}2 cells. Our results suggest that the Ln-5 {gamma}2 chain regulates the secretion of the {alpha}3 and {beta}3 subunits. More importantly, suppression of Ln-5 results in a phenotype that is representative of invasive tumor cells.

  1. Enhancement of varicella-zoster virus infection in cell lines expressing ORF4- or ORF62-encoded proteins.

    PubMed

    Schoonbroodt, S; Piette, J; Baudoux, L; Defechereux, P; Rentier, B; Merville, M P

    1996-08-01

    Varicella-Zoster virus (VZV) open reading frames 4 (ORF4) and 62 (ORF62) encode putative immediate early proteins (ORF4p and ORF62p, respectively) which are strong transactivators of other VZV genes and are involved in the very early stages of viral infection. ORF4p and ORF62p transactivate immediate-early and early gene promoters but have little or no effect on late gene promoters. To investigate the effect of ORF4p or ORF62p overexpression on the viral replication cycle, we constructed Vero cell lines expressing those genes under the control of the human cytomegalovirus major immediate-early promoter. VZV OKA infection of these stably transformed cell lines was followed-up using VZV glycoprotein E (gE) antigen quantification and virus titration. Upon serial passaging of infection in these cell lines expressing functionally active ORF4p or ORF62p, a 5- to 10-fold increase in viral gE antigen production was observed. Viral titers also demonstrated a 2- to 5-fold increase in viral production in these transformed cell lines. These results emphasize the role that both ORF4p and ORF62p play in enhancing the VZV replicative cycle.

  2. Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines

    PubMed Central

    Galetti, Maricla; Petronini, Pier Giorgio; Fumarola, Claudia; Cretella, Daniele; La Monica, Silvia; Bonelli, Mara; Cavazzoni, Andrea; Saccani, Francesca; Caffarra, Cristina; Andreoli, Roberta; Mutti, Antonio; Tiseo, Marcello; Ardizzoni, Andrea; Alfieri, Roberta R.

    2015-01-01

    Background BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism. Aim The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes. Methods and Results Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake. Conclusions Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells. PMID:26536031

  3. An intronic polymorphism associated with increased XRCC1 expression, reduced apoptosis and familial breast cancer.

    PubMed

    Bu, Dawei; Tomlinson, Gail; Lewis, Cheryl M; Zhang, Cindy; Kildebeck, Eric; Euhus, David M

    2006-10-01

    XRCC1 coordinates the activities of DNA polymerase-beta and DNA ligase for base excision repair of oxidative DNA damage. In addition, there is some evidence that XRCC1 is a negative regulator of apoptosis. Single nucleotide polymorphisms in XRCC1 have been inconsistently associated with breast cancer risk. We evaluated XRCC1 gene expression in breast cancer cell lines and carcinogen-induced apoptosis in benign breast epithelial cells in relation to XRCC1 genotypes. XRCC1 IVS10+141G>A was associated with increased expression of XRCC1 mRNA and protein, and reduced apoptosis in response to benzo-[a]-pyrene or ionizing radiation, but XRCC1 R399Q was not. These genotypes were also assessed in a clinic-based sample that included 190 breast cancer patients with a family history of breast cancer and 95 controls with no family history of breast cancer. Heterozygous XRCC1 IVS10+141G>A was associated with an increased breast cancer risk (O.R. = 1.7, 95% C.I. 1.016-2.827, P = 0.04) as was homozygous XRCC1 IVS10+141G>A (O.R. = 4.7, 95% C.I. 1.028-21.444, P = 0.03). XRCC1 R399Q was not associated with breast cancer (O.R. 1.00, 95% C.I. 0.61-1.64). The XRCC1 IVS10+141G>A locus is centered in a sequence that is nearly identical to the consensus binding site for the PLAG1 transcription factor. XRCC1 IVS10+141G>A is an intronic polymorphism that is associated with XRCC1 expression, apoptosis and familial breast cancer. It may occur within an intronic regulatory sequence. PMID:16596326

  4. The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

    PubMed

    Volpi, Chiara; Janni, Michela; Lionetti, Vincenzo; Bellincampi, Daniela; Favaron, Francesco; D'Ovidio, Renato

    2011-09-01

    Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

  5. CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression

    PubMed Central

    Liu, S-C; Chuang, S-M; Hsu, C-J; Tsai, C-H; Wang, S-W; Tang, C-H

    2014-01-01

    Connective tissue growth factor (CTGF, a.k.a. CCN2) is inflammatory mediator and abundantly expressed in osteoarthritis (OA). Angiogenesis is essential for OA progression. Here, we investigated the role of CTGF in vascular endothelial growth factor (VEGF) production and angiogenesis in OA synovial fibroblasts (OASFs). We showed that expression of CTGF and VEGF in synovial fluid were higher in OA patients than in controls. Directly applying CTGF to OASFs increased VEGF production then promoted endothelial progenitor cells tube formation and migration. CTGF induced VEGF by raising miR-210 expression via PI3K, AKT, ERK, and nuclear factor-κB (NF-κB)/ELK1 pathways. CTGF-mediating miR-210 upregulation repressed glycerol-3-phosphate dehydrogenase 1-like (GPD1L) expression and PHD activity and subsequently promoted hypoxia-inducible factor (HIF)-1α-dependent VEGF expression. Knockdown of CTGF decreased VEGF expression and abolished OASF-conditional medium-mediated angiogenesis in vitro as well as angiogenesis in chick chorioallantoic membrane and Matrigel-plug nude mice model in vivo. Taken together, our results suggest CTGF activates PI3K, AKT, ERK, and NF-κB/ELK1 pathway, leading to the upregulation of miR-210, contributing to inhibit GPD1L expression and prolyl hydroxylases 2 activity, promoting HIF-1α-dependent VEGF expression and angiogenesis in human synovial fibroblasts. PMID:25341039

  6. Lovastatin increases arachidonic acid levels and stimulates thromboxane synthesis in human liver and monocytic cell lines.

    PubMed Central

    Hrboticky, N; Tang, L; Zimmer, B; Lux, I; Weber, P C

    1994-01-01

    The effect of lovastatin (LOV), the inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, on linoleic acid (LA, 18:2n-6) metabolism was examined in human monocytic Mono Mac 6 (MM6) and hepatoma Hep G2 cells. The desaturation of LA was examined after LOV (72 h, 10 microM) or dimethylsulfoxide (LOV carrier, < 0.1%) and [14C]LA (last 18 h, 0.3 microCi, 5 microM). In both cell lines, LOV reduced the percentage of 14C label associated with LA and increased the percentage of label in the 20:4n-6 and the 22:5n-6 fractions. In Hep G2 but not MM6 cells, this effect was fully reversible by means of coincubation with mevalonic acid (500 microM), but not with cholesterol or lipoproteins. In both cell lines, the LOV-mediated increase in LA desaturation resulted in dose-dependent reductions of LA and elevations of AA in cellular phospholipids. The lipids secreted by LOV-treated Hep G2 cells were also enriched in arachidonic acid (AA). In the MM6 cells, LOV increased release of thromboxane upon stimulation with the calcium ionophore A23187. In summary, our findings of higher LA desaturation and AA enrichment of lipids secreted by the Hep G2 cells suggest that LOV treatment may increase the delivery of AA from the liver to extrahepatic tissues. The changes in membrane fatty acid composition can influence a variety of cellular functions, such as eicosanoid synthesis in monocytic cells. The mechanism appears to be related to the reduced availability of intermediates of cholesterogenesis. PMID:8282787

  7. Generation and characterization of transgenic plum lines expressing gafp-1 with the bul409 promoter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Gastrodia anti fungal protein (GAFP-1) is a mannose-binding lectin that can confer increased disease resistance in transgenic tobacco and plum. In all previously-generated transgenic lines, the gene was under the control of the 35SCaMV promoter. In this study, transgenic plum lines were create...

  8. Diversity of Reporter Expression Patterns in Transgenic Mouse Lines Targeting Corticotropin-Releasing Hormone-Expressing Neurons.

    PubMed

    Chen, Yuncai; Molet, Jenny; Gunn, Benjamin G; Ressler, Kerry; Baram, Tallie Z

    2015-12-01

    Transgenic mice, including lines targeting corticotropin-releasing factor (CRF or CRH), have been extensively employed to study stress neurobiology. These powerful tools are poised to revolutionize our understanding of the localization and connectivity of CRH-expressing neurons, and the crucial roles of CRH in normal and pathological conditions. Accurate interpretation of studies using cell type-specific transgenic mice vitally depends on congruence between expression of the endogenous peptide and reporter. If reporter expression does not faithfully reproduce native gene expression, then effects of manipulating unintentionally targeted cells may be misattributed. Here, we studied CRH and reporter expression patterns in 3 adult transgenic mice: Crh-IRES-Cre;Ai14 (tdTomato mouse), Crfp3.0CreGFP, and Crh-GFP BAC. We employed the CRH antiserum generated by Vale after validating its specificity using CRH-null mice. We focused the analyses on stress-salient regions, including hypothalamus, amygdala, bed nucleus of the stria terminalis, and hippocampus. Expression patterns of endogenous CRH were consistent among wild-type and transgenic mice. In tdTomato mice, most CRH-expressing neurons coexpressed the reporter, yet the reporter identified a few non-CRH-expressing pyramidal-like cells in hippocampal CA1 and CA3. In Crfp3.0CreGFP mice, coexpression of CRH and the reporter was found in central amygdala and, less commonly, in other evaluated regions. In Crh-GFP BAC mice, the large majority of neurons expressed either CRH or reporter, with little overlap. These data highlight significant diversity in concordant expression of reporter and endogenous CRH among 3 available transgenic mice. These findings should be instrumental in interpreting important scientific findings emerging from the use of these potent neurobiological tools. PMID:26402844

  9. Ectopic expression of anthocyanin 5-o-glucosyltransferase in potato tuber causes increased resistance to bacteria.

    PubMed

    Lorenc-Kukuła, Katarzyna; Jafra, Sylwia; Oszmiański, Jan; Szopa, Jan

    2005-01-26

    The principal goal of this paper was to investigate the significance of anthocyanin 5-O-glucosyltransferase (5-UGT) for potato tuber metabolism. The ectopic expression of a 5-UGT cDNA in the tuber improved the plant's defense against pathogen infection. The resistance of transgenic lines against Erwinia carotovora subsp. carotovora was about 2-fold higher than for nontransformed plants. In most cases the pathogen resistance was accompanied by a significant increase in tuber yield. To investigate the molecular basis of transgenic potato resistance, metabolic profiling of the plant was performed. In tuber extracts, the anthocyanin 3,5-O-substituted level was significantly increased when compared to that of the control plant. Of six anthocyanin compounds identified, the highest quantity for pelargonidin 3-rutinoside-5-glucoside acylated with p-coumaric acid and peonidin 3-rutinoside-5-glucoside acylated with p-coumaric acid was detected. A significant increase in starch and a decrease in sucrose level in transgenic tubers have been detected. The level of all other metabolites (amino acids, organic acids, polyamines, and fatty acids) was quite the same as in nontransformants. The plant resistance to bacterial infection correlates with anthocyanin content and sucrose level. The properties of recombinant glucosyltransferase were analyzed in in vitro experiments. The enzyme kinetics and its biochemical properties were similar to those from other sources.

  10. Increase in intracellular PGE2 induces apoptosis in Bax-expressing colon cancer cell

    PubMed Central

    2011-01-01

    Background NSAIDs exhibit protective properties towards some cancers, especially colon cancer. Yet, it is not clear how they play their protective role. PGE2 is generally shown as the only target of the NSAIDs anticancerous activity. However, PGE2 known targets become more and more manifold, considering both the molecular pathways involved and the target cells in the tumour. The role of PGE2 in tumour progression thus appears complex and multipurpose. Methods To gain understanding into the role of PGE2 in colon cancer, we focused on the activity of PGE2 in apoptosis in colon cancer cell lines. Results We observed that an increase in intracellular PGE2 induced an apoptotic cell death, which was dependent on the expression of the proapoptotic protein Bax. This increase was induced by increasing PGE2 intracellular concentration, either by PGE2 microinjection or by the pharmacological inhibition of PGE2 exportation and enzymatic degradation. Conclusions We present here a new sight onto PGE2 in colon cancer cells opening the way to a new prospective therapeutic strategy in cancer, alternative to NSAIDs. PMID:21524287

  11. Matrigel Basement Membrane Matrix influences expression of microRNAs in cancer cell lines

    SciTech Connect

    Price, Karina J.; Tsykin, Anna; Giles, Keith M.; Sladic, Rosemary T.; Epis, Michael R.; Ganss, Ruth; Goodall, Gregory J.; Leedman, Peter J.

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Matrigel alters cancer cell line miRNA expression relative to culture on plastic. Black-Right-Pointing-Pointer Many identified Matrigel-regulated miRNAs are implicated in cancer. Black-Right-Pointing-Pointer miR-1290, -210, -32 and -29b represent a Matrigel-induced miRNA signature. Black-Right-Pointing-Pointer miR-32 down-regulates Integrin alpha 5 (ITGA5) mRNA. -- Abstract: Matrigel is a medium rich in extracellular matrix (ECM) components used for three-dimensional cell culture and is known to alter cellular phenotypes and gene expression. microRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and have roles in cancer. While miRNA profiles of numerous cell lines cultured on plastic have been reported, the influence of Matrigel-based culture on cancer cell miRNA expression is largely unknown. This study investigated the influence of Matrigel on the expression of miRNAs that might facilitate ECM-associated cancer cell growth. We performed miRNA profiling by microarray using two colon cancer cell lines (SW480 and SW620), identifying significant differential expression of miRNAs between cells cultured in Matrigel and on plastic. Many of these miRNAs have previously been implicated in cancer-related processes. A common Matrigel-induced miRNA signature comprised of up-regulated miR-1290 and miR-210 and down-regulated miR-29b and miR-32 was identified using RT-qPCR across five epithelial cancer cell lines (SW480, SW620, HT-29, A549 and MDA-MB-231). Experimental modulation of these miRNAs altered expression of their known target mRNAs involved in cell adhesion, proliferation and invasion, in colon cancer cell lines. Furthermore, ITGA5 was identified as a novel putative target of miR-32 that may facilitate cancer cell interactions with the ECM. We propose that culture of cancer cell lines in Matrigel more accurately recapitulates miRNA expression and function in cancer than culture on plastic and thus is a

  12. Microarray-based detection and expression analysis of ABC and SLC transporters in drug-resistant ovarian cancer cell lines.

    PubMed

    Januchowski, Radosław; Zawierucha, Piotr; Andrzejewska, Małgorzata; Ruciński, Marcin; Zabel, Maciej

    2013-04-01

    Multiple drug resistance of cancer cells is multifactorial. A microarray technique may provide information about new candidate genes playing a role in drug resistance. Drug membrane transporters from ABC and SLC families play a main role in this phenomenon. This study demonstrates alterations in ABC and SLC gene expression levels in methotrexate, cisplatin, doxorubicin, vincristine, topotecan and paclitaxel-resistant variant of W1 ovarian cancer cell line. Resistant W1 cell lines were derived by stepwise selection of cells in increasing concentration of drugs. Affymetrix GeneChip(®) Human Genome U219 Array Strip was used for hybridizations. Statistical significance was determined by independent sample t-test. The genes having altered expression levels in drug-resistant sublines were selected and filtered by scater plot. Genes up/downregulated more than threefolds were selected and listed. Among ABC genes, seven were upregulated and three were downregulated. Three genes: ABCB1, ABCB4 and ABCG2 were upregulated very significantly (over tenfold). One ABCA8 was significantly downregulated. Among 38 SLC genes, 18 were upregulated, 16 were downregulated and four were up- or downregulated dependent on the cell line. Expression of 10 SLC genes was changed very significantly (over tenfold). Four genes were significantly increased: SLC6A1, SLC9A2, SLC12A1, SLC16A6 and six genes were significantly decreased: SLC2A14, SLC7A3, SLC7A8, SLC7A11, SLC16A14, SLC38A9. Based on the expression profiles, our results provide a preliminary insight into the relationship between drug resistance and expression of membrane transporters involved in drug resistance. Correlation of specific drug transporter with drug resistance requires further analysis.

  13. "Fluorescent Cell Chip" for immunotoxicity testing: development of the c-fos expression reporter cell lines.

    PubMed

    Trzaska, Dominika; Zembek, Patrycja; Olszewski, Maciej; Adamczewska, Violetta; Ullerås, Erik; Dastych, Jarosław

    2005-09-01

    The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals.

  14. "Life without line dancing and the other activities would be too dreadful to imagine": an increase in social activity for older women.

    PubMed

    Nadasen, Krishnavelli

    2008-01-01

    The literature on aging is replete with the positive effects of physical exercise on the well-being of older adults. Some suggest, however, that the potential impact of social activities has not received adequate attention, and others note the importance of distinguishing between the physical and nonphysical impact of these activities. This study investigated whether line dancing, a physical activity, led to an increase in social activity. Thirty women over the age of 60 were interviewed to discover how line dancing affected them. Content analysis of the interviews helped identify various themes indicating that line dancing enabled these women to expand their repertoire of social activity, leading to positive reinforcements such as further community involvement, charitable work, inclusion in national sports events, self-expression, and personal development. The impact of line dancing plainly goes beyond the perceived physical benefits.

  15. Differential Expression of OCT4 Pseudogenes in Pluripotent and Tumor Cell Lines

    PubMed Central

    Poursani, Ensieh M.; Mohammad Soltani, Bahram; Mowla, Seyed Javad

    2016-01-01

    Objective The human OCT4 gene, the most important pluripotency marker, can generate at least three different transcripts (OCT4A, OCT4B, and OCT4B1) by alternative splicing. OCT4A is the main isoform responsible for the stemness property of embryonic stem (ES) cells. There also exist eight processed OCT4 pseudogenes in the human genome with high homology to the OCT4A, some of which are transcribed in various cancers. Recent conflicting reports on OCT4 expression in tumor cells and tissues emphasize the need to discriminate the expression of OCT4A from other variants as well as OCT4 pseudogenes. Materials and Methods In this experimental study, DNA sequencing confirmed the authenticity of transcripts of OCT4 pseudogenes and their expression patterns were investigated in a panel of different human cell lines by reverse transcription-polymerase chain reaction (RT-PCR). Results Differential expression of OCT4 pseudogenes in various human cancer and pluripotent cell lines was observed. Moreover, the expression pattern of OCT4-pseudogene 3 (OCT4-pg3) followed that of OCT4A during neural differentiation of the pluripotent cell line of NTERA-2 (NT2). Although OCT4-pg3 was highly expressed in undifferentiated NT2 cells, its expression was rapidly down-regulated upon induction of neural differentiation. Analysis of protein expression of OCT4A, OCT4-pg1, OCT4-pg3, and OCT4-pg4 by Western blotting indicated that OCT4 pseudogenes cannot produce stable proteins. Consistent with a newly proposed competitive role of pseudogene microRNA docking sites, we detected miR-145 binding sites on all transcripts of OCT4 and OCT4 pseudogenes. Conclusion Our study suggests a potential coding-independent function for OCT4 pseudogenes during differentiation or tumorigenesis. PMID:27054116

  16. Lineage-restricted expression of homeobox-containing genes in human hematopoietic cell lines.

    PubMed

    Shen, W F; Largman, C; Lowney, P; Corral, J C; Detmer, K; Hauser, C A; Simonitch, T A; Hack, F M; Lawrence, H J

    1989-11-01

    We investigated the role of homeobox-containing genes in human hematopoiesis because homeobox genes (i) control cell fate in the Drosophila embryo, (ii) are expressed in specific patterns in human embryos, and (iii) appear to function as transcription factors that control cell phenotype in other mammalian organs. Using four homeobox probes from the HOX2 locus and a previously undescribed homeobox cDNA (PL1), we screened mRNAs from 18 human leukemic cell lines representing erythroid, myeloid, and T- and B-cell lineages. Complex patterns of lineage-restricted expression are observed: some are restricted to a single lineage, while others are expressed in multiple lineages. No single homeobox gene is expressed in all types of hematopoietic cells, but each cell type exhibits homeobox gene expression. HOX2.2 and -2.3 homeobox-containing cDNAs were cloned from an erythroleukemia cell (HEL) cDNA library, while the homeobox cDNA PL1 was isolated from a monocytic cell (U-937) library. Differentiation of HEL and K-562 cells with various inducers results in modulation of specific homeobox transcripts. In addition, HOX2.2 is expressed in normal bone marrow cells. We have demonstrated (i) lineage-restricted expression of five homeobox genes in erythroid and monocytic cell lines; (ii) expression of additional homeobox genes in other cell lineages (HL-60 and lymphoid cells); (iii) expression of one homeobox gene in normal marrow cells; and (iv) modulation of expression during differentiation. These data suggest that these genes play a role in human hematopoietic development and lineage commitment.

  17. Lineage-restricted expression of homeobox-containing genes in human hematopoietic cell lines.

    PubMed Central

    Shen, W F; Largman, C; Lowney, P; Corral, J C; Detmer, K; Hauser, C A; Simonitch, T A; Hack, F M; Lawrence, H J

    1989-01-01

    We investigated the role of homeobox-containing genes in human hematopoiesis because homeobox genes (i) control cell fate in the Drosophila embryo, (ii) are expressed in specific patterns in human embryos, and (iii) appear to function as transcription factors that control cell phenotype in other mammalian organs. Using four homeobox probes from the HOX2 locus and a previously undescribed homeobox cDNA (PL1), we screened mRNAs from 18 human leukemic cell lines representing erythroid, myeloid, and T- and B-cell lineages. Complex patterns of lineage-restricted expression are observed: some are restricted to a single lineage, while others are expressed in multiple lineages. No single homeobox gene is expressed in all types of hematopoietic cells, but each cell type exhibits homeobox gene expression. HOX2.2 and -2.3 homeobox-containing cDNAs were cloned from an erythroleukemia cell (HEL) cDNA library, while the homeobox cDNA PL1 was isolated from a monocytic cell (U-937) library. Differentiation of HEL and K-562 cells with various inducers results in modulation of specific homeobox transcripts. In addition, HOX2.2 is expressed in normal bone marrow cells. We have demonstrated (i) lineage-restricted expression of five homeobox genes in erythroid and monocytic cell lines; (ii) expression of additional homeobox genes in other cell lineages (HL-60 and lymphoid cells); (iii) expression of one homeobox gene in normal marrow cells; and (iv) modulation of expression during differentiation. These data suggest that these genes play a role in human hematopoietic development and lineage commitment. Images PMID:2573064

  18. Genetic instability in lymphoblastoid cell lines expressing biallelic and monoallelic variants in the human MUTYH gene.

    PubMed

    Grasso, Francesca; Giacomini, Elisa; Sanchez, Massimo; Degan, Paolo; Gismondi, Viviana; Mazzei, Filomena; Varesco, Liliana; Viel, Alessandra; Bignami, Margherita

    2014-07-15

    The MUTYH DNA glycosylase counteracts mutagenesis by removing adenine misincorporated opposite DNA 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated polyposis (MAP). The impact on genetic instability of the p.Tyr179Cys and p.Arg245His MUTYH variants was evaluated in lymphoblastoid cell lines (LCLs) derived from MAP patients and their relatives in comparison to wild-type LCLs. No difference in MUTYH expression was identified between wild type and LCLs with the p.Tyr179Cys, while the p.Arg245His mutation was associated with an unstable MUTYH protein. LCLs homozygous for the p.Tyr179Cys or the p.Arg245His variant contained increased DNA 8-oxodG levels and exhibited a mutator phenotype at the PIG-A gene. The extent of the increased spontaneous mutation frequency was 3-fold (range 1.6- to 4.6-fold) in four independent LCLs carrying the p.Tyr179Cys variant, while a larger increase (6-fold) was observed in two p.Arg245His LCLs. A similar hypermutability and S-phase delay following treatment with KBrO3 was observed in LCLs homozygous for either variant. When genetic instability was investigated in monoallelic p.Arg245His carriers, mutant frequencies showed an increase which is intermediate between wild-type and homozygous cells, whereas the mutator effect in heterozygous p.Tyr179Cys LCLs was similar to that in homozygotes. These findings indicate that the type of MUTYH mutation can affect the extent of genome instability associated with MUTYH inactivation. In addition, the mild spontaneous mutator phenotype observed in monoallelic carriers highlights the biological importance of this gene in the protection of the genome against endogenous DNA damage.

  19. Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines

    PubMed Central

    Ghosheh, Nidal; Olsson, Björn; Edsbagge, Josefina; Küppers-Munther, Barbara; Van Giezen, Mariska; Asplund, Annika; Andersson, Tommy B.; Björquist, Petter; Carén, Helena; Simonsson, Stina; Sartipy, Peter; Synnergren, Jane

    2016-01-01

    Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models. PMID:26949401

  20. An improved HAdV-41 E1B55K-expressing 293 cell line for packaging fastidious adenovirus.

    PubMed

    Zou, Xiao-Hui; Xiao, Xia; Chen, Duo-Ling; Li, Ze-Liang; Song, Jing-Dong; Wang, Min; Qu, Jian-Guo; Lu, Zhuo-Zhuang; Hung, Tao

    2011-08-01

    Human adenovirus type 41 (HAdV-41) is difficult to cultivate under laboratory conditions. Tripartite leader sequence (TPL) of HAdV-41 was cloned, inserted into eukaryotic expression plasmid, and used to establish a HAdV-41 E1B55K-transduced cell line (293TE7). HAdV-41 E1B55K was expressed more abundantly in 293TE7 than in 293E12, an HAdV-41 E1B55K-expressing cell line developed previously. After being infected with E1-deleted HAdV-41 vector (HAdV-41-GFP), 293TE7 synthesized more viral genomic DNA and structural proteins, which led ultimately to a significant increase of the yield of progeny viruses. Typically, 293TE7 produced progeny viruses 3-15 times more than 293E12 did, depending on the amount of seed viruses and culture time. These data demonstrated that 293TE7 was an effective packaging cell line, and implied its application for wild-type HAdV-41 isolation, HAdV-41 virological study and recombinant HAdV-41 construction. PMID:21601594

  1. Expression of the Pokemon proto-oncogene in nasopharyngeal carcinoma cell lines and tissues.

    PubMed

    Jiao, Wei; Liu, Fei; Tang, Feng-Zhu; Lan, Jiao; Xiao, Rui-Ping; Chen, Xing-Zhou; Ye, Hui-Lan; Cai, Yong-Lin

    2013-01-01

    To study the differentiated expression of the proto-oncogene Pokemon in nasopharyngeal carcinoma (NPC) cell lines and tissues, mRNA and protein expression levels of CNE1, CNE2, CNE3 and C666-1 were detected separately by reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and Western-blotting. The immortalized nasopharyngeal epithelial cell line NP69 was used as a control. The Pokemon protein expression level in biopsy specimens from chronic rhinitis patients and undifferentiated non keratinizing NPC patients was determined by Western-blotting and arranged from high to low: C666-1>CNE1>CNE2> CNE3>NP69. The Pokemon mRNA expression level was also arranged from high to low: CNE1>CNE2>NP69>C666-1>CNE3. Pokemon expression of NP69 and C666-1 obviously varied from mRNA to protein. The Pokemon protein level of NPC biopsy specimens was obviously higher than in chronic rhinitis. The data suggest that high Pokemon protein expression is closely associated with undifferentiated non-keratinizing NPC and may provide useful information for NPC molecular target therapy. PMID:24377524

  2. Expression of the Pokemon proto-oncogene in nasopharyngeal carcinoma cell lines and tissues.

    PubMed

    Jiao, Wei; Liu, Fei; Tang, Feng-Zhu; Lan, Jiao; Xiao, Rui-Ping; Chen, Xing-Zhou; Ye, Hui-Lan; Cai, Yong-Lin

    2013-01-01

    To study the differentiated expression of the proto-oncogene Pokemon in nasopharyngeal carcinoma (NPC) cell lines and tissues, mRNA and protein expression levels of CNE1, CNE2, CNE3 and C666-1 were detected separately by reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and Western-blotting. The immortalized nasopharyngeal epithelial cell line NP69 was used as a control. The Pokemon protein expression level in biopsy specimens from chronic rhinitis patients and undifferentiated non keratinizing NPC patients was determined by Western-blotting and arranged from high to low: C666-1>CNE1>CNE2> CNE3>NP69. The Pokemon mRNA expression level was also arranged from high to low: CNE1>CNE2>NP69>C666-1>CNE3. Pokemon expression of NP69 and C666-1 obviously varied from mRNA to protein. The Pokemon protein level of NPC biopsy specimens was obviously higher than in chronic rhinitis. The data suggest that high Pokemon protein expression is closely associated with undifferentiated non-keratinizing NPC and may provide useful information for NPC molecular target therapy.

  3. The effect of freeze-thaw cycles on gene expression levels in lymphoblastoid cell lines.

    PubMed

    Çalışkan, Minal; Pritchard, Jonathan K; Ober, Carole; Gilad, Yoav

    2014-01-01

    Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene expression. We report a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene expression levels in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. We found that previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that while insight drawn from gene regulatory studies in mature LCLs may generally not be affected by the artificial nature of the LCL model system, many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures.

  4. The Effect of Freeze-Thaw Cycles on Gene Expression Levels in Lymphoblastoid Cell Lines

    PubMed Central

    Çalışkan, Minal; Pritchard, Jonathan K.; Ober, Carole; Gilad, Yoav

    2014-01-01

    Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene expression. We report a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene expression levels in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. We found that previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that while insight drawn from gene regulatory studies in mature LCLs may generally not be affected by the artificial nature of the LCL model system, many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures. PMID:25192014

  5. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    SciTech Connect

    Zhou Huimin; Jia Li; Wang Shujing; Wang Hongmei; Chu Haiying; Hu Yichuan; Cao Jun; Zhang Jianing . E-mail: jnzhang@dlmedu.edu.cn

    2006-06-23

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression.

  6. Increased susceptibility to ethylmercury-induced mitochondrial dysfunction in a subset of autism lymphoblastoid cell lines.

    PubMed

    Rose, Shannon; Wynne, Rebecca; Frye, Richard E; Melnyk, Stepan; James, S Jill

    2015-01-01

    The association of autism spectrum disorders with oxidative stress, redox imbalance, and mitochondrial dysfunction has become increasingly recognized. In this study, extracellular flux analysis was used to compare mitochondrial respiration in lymphoblastoid cell lines (LCLs) from individuals with autism and unaffected controls exposed to ethylmercury, an environmental toxin known to deplete glutathione and induce oxidative stress and mitochondrial dysfunction. We also tested whether pretreating the autism LCLs with N-acetyl cysteine (NAC) to increase glutathione concentrations conferred protection from ethylmercury. Examination of 16 autism/control LCL pairs revealed that a subgroup (31%) of autism LCLs exhibited a greater reduction in ATP-linked respiration, maximal respiratory capacity, and reserve capacity when exposed to ethylmercury, compared to control LCLs. These respiratory parameters were significantly elevated at baseline in the ethylmercury-sensitive autism subgroup as compared to control LCLs. NAC pretreatment of the sensitive subgroup reduced (normalized) baseline respiratory parameters and blunted the exaggerated ethylmercury-induced reserve capacity depletion. These findings suggest that the epidemiological link between environmental mercury exposure and an increased risk of developing autism may be mediated through mitochondrial dysfunction and support the notion that a subset of individuals with autism may be vulnerable to environmental influences with detrimental effects on development through mitochondrial dysfunction.

  7. Increased Susceptibility to Ethylmercury-Induced Mitochondrial Dysfunction in a Subset of Autism Lymphoblastoid Cell Lines

    PubMed Central

    Wynne, Rebecca; Frye, Richard E.; Melnyk, Stepan; James, S. Jill

    2015-01-01

    The association of autism spectrum disorders with oxidative stress, redox imbalance, and mitochondrial dysfunction has become increasingly recognized. In this study, extracellular flux analysis was used to compare mitochondrial respiration in lymphoblastoid cell lines (LCLs) from individuals with autism and unaffected controls exposed to ethylmercury, an environmental toxin known to deplete glutathione and induce oxidative stress and mitochondrial dysfunction. We also tested whether pretreating the autism LCLs with N-acetyl cysteine (NAC) to increase glutathione concentrations conferred protection from ethylmercury. Examination of 16 autism/control LCL pairs revealed that a subgroup (31%) of autism LCLs exhibited a greater reduction in ATP-linked respiration, maximal respiratory capacity, and reserve capacity when exposed to ethylmercury, compared to control LCLs. These respiratory parameters were significantly elevated at baseline in the ethylmercury-sensitive autism subgroup as compared to control LCLs. NAC pretreatment of the sensitive subgroup reduced (normalized) baseline respiratory parameters and blunted the exaggerated ethylmercury-induced reserve capacity depletion. These findings suggest that the epidemiological link between environmental mercury exposure and an increased risk of developing autism may be mediated through mitochondrial dysfunction and support the notion that a subset of individuals with autism may be vulnerable to environmental influences with detrimental effects on development through mitochondrial dysfunction. PMID:25688267

  8. Global gene expression in pseudomyxoma peritonei, with parallel development of two immortalized cell lines.

    PubMed

    Roberts, Darren L; O'Dwyer, Sarah T; Stern, Peter L; Renehan, Andrew G

    2015-05-10

    Pseudomyxoma peritonei (PMP) is a rare tumor of appendiceal origin. Treatment is major cytoreductive surgery but morbidity is high. PMP is considered chemo-resistant; its molecular biology is understudied; and presently, there is no platform for pre-clinical drug testing. Here, we performed exon array analysis from laser micro-dissected PMP tissue and normal colonic epithelia. The array analysis identified 27 up-regulated and 34 down-regulated genes: candidate up-regulated genes included SLC16A4, DSC3, Aldolase B, EPHX4, and ARHGAP24; candidate down-regulated genes were MS4A12, TMIGD1 and Caspase-5. We confirmed differential expression of the candidate genes and their protein products using in-situ hybridization and immuno-histochemistry. In parallel, we established two primary PMP cell lines, N14A and N15A, and immortalized with an SV40 T-antigen lentiviral vector. We cross-checked for expression of the candidate genes (from the array analyses) using qPCR in the cell lines and demonstrated that the gene profiles were distinct from those of colorectal tumor libraries and commonly used colon cell lines. N14A and N15A were responsiveness to mitomycin and oxaliplatin. This study characterizes global gene expression in PMP, and the parallel development of the first immortalized PMP cell lines; fit for pre-clinical testing and PMP oncogene discovery.

  9. Thrombopoietin receptor expression in human cancer cell lines and primary tissues.

    PubMed

    Columbyova, L; Loda, M; Scadden, D T

    1995-08-15

    c-mpl is the receptor for the recently identified megakaryocyte growth and differentiation factor thrombopoietin. Thrombopoietin has been shown to be capable of raising platelet counts in animals and is about to enter clinical trials in humans. In anticipation of its likely use in the care of patients receiving cancer chemotherapy, we evaluated the expression of human c-mpl by reverse transcription PCR on 39 human cell lines and 20 primary human tissue samples derived from both normal and malignant sources. c-mpl transcripts were found in all megakaryocytic cell lines tested (CMK, CMK-2B, CMK-2D, SO, and DAMI), the CD34+ leukemia cell line KMT-2, and a hepatocellular carcinoma cell line (Hep3B). Among primary tissues, fetal liver cells and brain had detectable levels of c-mpl message, and among primary tumors, none were found to express c-mpl. These data support the conclusion that c-mpl has restricted expression that is primarily, but not exclusively, related to megakaryocytopoiesis. These observations suggest that thrombopoietin is unlikely to have direct effects on other malignant or normal tissue should it have a clinical role in the treatment of chemotherapy-induced thrombocytopenia. PMID:7627956

  10. Global gene expression in pseudomyxoma peritonei, with parallel development of two immortalized cell lines

    PubMed Central

    Roberts, Darren L.; O'Dwyer, Sarah T.; Stern, Peter L.; Renehan, Andrew G.

    2015-01-01

    Pseudomyxoma peritonei (PMP) is a rare tumor of appendiceal origin. Treatment is major cytoreductive surgery but morbidity is high. PMP is considered chemo-resistant; its molecular biology is understudied; and presently, there is no platform for pre-clinical drug testing. Here, we performed exon array analysis from laser micro-dissected PMP tissue and normal colonic epithelia. The array analysis identified 27 up-regulated and 34 down-regulated genes: candidate up-regulated genes included SLC16A4, DSC3, Aldolase B, EPHX4, and ARHGAP24; candidate down-regulated genes were MS4A12, TMIGD1 and Caspase-5. We confirmed differential expression of the candidate genes and their protein products using in-situ hybridization and immuno-histochemistry. In parallel, we established two primary PMP cell lines, N14A and N15A, and immortalized with an SV40 T-antigen lentiviral vector. We cross-checked for expression of the candidate genes (from the array analyses) using qPCR in the cell lines and demonstrated that the gene profiles were distinct from those of colorectal tumor libraries and commonly used colon cell lines. N14A and N15A were responsiveness to mitomycin and oxaliplatin. This study characterizes global gene expression in PMP, and the parallel development of the first immortalized PMP cell lines; fit for pre-clinical testing and PMP oncogene discovery. PMID:25929336

  11. Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Ye, C.; Vassilev, P. M.; Sanders, J. L.; Brown, E. M.

    2001-01-01

    We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

  12. Increased gastrin gene expression provides a physiological advantage to mice under hypoxic conditions.

    PubMed

    Laval, Marie; Baldwin, Graham S; Shulkes, Arthur; Marshall, Kathryn M

    2015-01-15

    Hypoxia, or a low concentration of O2, is encountered in humans undertaking activities such as mountain climbing and scuba diving and is important pathophysiologically as a limiting factor in tumor growth. Although data on the interplay between hypoxia and gastrins are limited, gastrin expression is upregulated by hypoxia in gastrointestinal cancer cell lines, and gastrins counterbalance hypoxia by stimulating angiogenesis in vitro and in vivo. The aim of this study was to determine if higher concentrations of the gastrin precursor progastrin are protective against hypoxia in vivo. hGAS mice, which overexpress progastrin in the liver, and mice of the corresponding wild-type FVB/N strain were exposed to normoxia or hypoxia. Iron status was assessed by measurement of serum iron parameters, real-time PCR for mRNAs encoding critical iron regulatory proteins, and Perls' stain and atomic absorption spectrometry for tissue iron concentrations. FVB/N mice lost weight at a faster rate and had higher sickness scores than hGAS mice exposed to hypoxia. Serum iron levels were lower in hGAS than FVB/N mice and decreased further when the animals were exposed to hypoxia. The concentration of iron in the liver was strikingly lower in hGAS than FVB/N mice. We conclude that increased circulating concentrations of progastrin provide a physiological advantage against systemic hypoxia in mice, possibly by increasing the availability of iron stores. This is the first report of an association between progastrin overexpression, hypoxia, and iron homeostasis.

  13. Cell line specific modulation of connexin43 expression after exposure to ionizing radiation.

    PubMed

    Banaz-Yaşar, Ferya; Tischka, Rabea; Iliakis, George; Winterhager, Elke; Gellhaus, Alexandra

    2005-01-01

    Gap junctional intercellular communication plays a significant role in mediating radiation-induced bystander effects. However, the level of Cx43 itself is influenced by ionizing radiation, which could modify the bystander effect. Here we have investigated several cell lines for the modulation of Cx43 expression 24 h after irradiation with 5 Gy X-rays. The mouse endothelial cell line bEnd3 revealed a significantly elevated level of Cx43 already 15 min after exposure to X-rays, whereas human hybrid endothelial cells (EA.hy926) exhibited a transient downregulation of Cx43 mRNA. No obvious changes in the communication properties of the different cell lines could be observed after irradiation. The communication-deficient malignant human trophoblast cell line Jeg3 stably transfected with Cx43 did not reveal any induction of endogenous nor alteration in the exogenous Cx43 transcript level upon exposure to 5 Gy. Taken together, our data show a cell line specific modulation of Cx43 expression after exposure to X-rays.

  14. Cell line specific modulation of connexin43 expression after exposure to ionizing radiation.

    PubMed

    Banaz-Yaşar, Ferya; Tischka, Rabea; Iliakis, George; Winterhager, Elke; Gellhaus, Alexandra

    2005-01-01

    Gap junctional intercellular communication plays a significant role in mediating radiation-induced bystander effects. However, the level of Cx43 itself is influenced by ionizing radiation, which could modify the bystander effect. Here we have investigated several cell lines for the modulation of Cx43 expression 24 h after irradiation with 5 Gy X-rays. The mouse endothelial cell line bEnd3 revealed a significantly elevated level of Cx43 already 15 min after exposure to X-rays, whereas human hybrid endothelial cells (EA.hy926) exhibited a transient downregulation of Cx43 mRNA. No obvious changes in the communication properties of the different cell lines could be observed after irradiation. The communication-deficient malignant human trophoblast cell line Jeg3 stably transfected with Cx43 did not reveal any induction of endogenous nor alteration in the exogenous Cx43 transcript level upon exposure to 5 Gy. Taken together, our data show a cell line specific modulation of Cx43 expression after exposure to X-rays. PMID:16531320

  15. Over-expression of OsPTR6 in rice increased plant growth at different nitrogen supplies but decreased nitrogen use efficiency at high ammonium supply.

    PubMed

    Fan, Xiaorong; Xie, Dan; Chen, Jingguang; Lu, Haiyan; Xu, Yanling; Ma, Cui; Xu, Guohua

    2014-10-01

    Nitrogen (N) plays a critical role in plant growth and productivity and PTR/NRT1 transporters are critical for rice growth. In this study, OsPTR6, a PTR/NRT1 transporter, was over-expressed in the Nipponbare rice cultivar by Agrobacterium tumefaciens transformation using the ubiquitin (Ubi) promoter. Three single-copy T2 generation transgenic lines, named OE1, OE5 and OE6, were produced and subjected to hydroponic growth experiments in different nitrogen treatments. The results showed the plant height and biomass of the over-expression lines were increased, and plant N accumulation and glutamine synthetase (GS) activities were enhanced at 5.0mmol/L NH4(+) and 2.5mmol/L NH4NO3. The expression of OsATM1 genes in over-expression lines showed that the OsPTR6 over expression increased OsAMT1.1, OsATM1.2 and OsAMT1.3 expression at 0.2 and 5.0mmol/L NH4(+) and 2.5mmol/L NH4NO3. However, nitrogen utilisation efficiency (NUE) was decreased at 5.0mmol/LNH4(+). These data suggest that over-expression of the OsPTR6 gene could increase rice growth through increasing ammonium transporter expression and glutamine synthetase activity (GSA), but decreases nitrogen use efficiency under conditions of high ammonium supply.

  16. Differential expression of the ufo/axl oncogene in human leukemia-lymphoma cell lines.

    PubMed

    Challier, C; Uphoff, C C; Janssen, J W; Drexler, H G

    1996-05-01

    The ufo protein (also termed axl) is a member of a new family of receptor tyrosine kinases and is encoded by a transforming gene that was initially isolated from primary human myeloid leukemia cells by DNA-mediated transformation of NIH/3T3 cells. The ligand, Gas6, a protein S-related molecule lacking any known function yet, has recently been identified. We report the expression pattern of ufo mRNA in a panel of 76 human continuous leukemia-lymphoma cell lines. The gene was not expressed in cell lines derived from lymphoid malignancies (n=28), but transcription was seen in 3/11 myeloid, 0/6 monocytic, 9/13 erythroid and 11/18 megakaryocytic cell lines. Several cell lines were treated with phorbol ester leading to significant upregulation of the ufo message in constitutively positive cells. An apparent ufo mRNA overexpression was not found in any of the positive leukemia cell lines, but was identified in the drug-resistant subclones of the cervix carcinoma cell line HeLa. Southern blot analysis of restriction enzyme-digested genomic DNA did not provide evidence for gene amplification, but the HeLa subclones showed banding patterns suggestive of gene rearrangement. Two main ufo mRNA bands of 3.2 and 5.0 kb were identified; no differences in the half-lives (t1/2 = 2.5 h) of these two mRNA species could be identified. In summary, ufo, representing a novel type of receptor tyrosine kinase, is expressed solely in myeloid and erythro-megakaryocytic leukemias but not in lymphoid malignancies. These and previous data suggest an involvement of the ufo receptor tyrosine kinase in normal and malignant myelopoiesis; however, its exact role, if any, and mode of operation in leukemogenesis remains to be determined.

  17. Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

    PubMed Central

    Haggerty, Timothy J.; Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Pandolfi, Franco; Kurnick, James T.

    2014-01-01

    In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer. PMID:25503774

  18. Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants

    PubMed Central

    Gottschalk, Laura B.; Vecchio-Pagan, Briana; Sharma, Neeraj; Han, Sangwoo T.; Franca, Arianna; Wohler, Elizabeth S.; Batista, Denise A.S.; Goff, Loyal A.; Cutting, Garry R.

    2016-01-01

    Background Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells. Methods A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o−). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues. Results Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways. Conclusion CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context. PMID:26694805

  19. Expression, localization, and regulation of NOS in human mast cell lines: effects on leukotriene production.

    PubMed

    Gilchrist, Mark; McCauley, Scott D; Befus, A Dean

    2004-07-15

    Nitric oxide (NO) is a potent radical produced by nitric oxide synthase (NOS) and has pleiotrophic activities in health and disease. As mast cells (MCs) play a central role in both homeostasis and pathology, we investigated NOS expression and NO production in human MC populations. Endothelial NOS (eNOS) was ubiquitously expressed in both human MC lines and skin-derived MCs, while neuronal NOS (nNOS) was variably expressed in the MC populations studied. The inducible (iNOS) isoform was not detected in human MCs. Both growth factor-independent (HMC-1) and -dependent (LAD 2) MC lines showed predominant nuclear eNOS protein localization, with weaker cytoplasmic expression. nNOS showed exclusive cytoplasmic localization in HMC-1. Activation with Ca(2+) ionophore (A23187) or IgE-anti-IgE induced eNOS phosphorylation and translocation to the nucleus and nuclear and cytoplasmic NO formation. eNOS colocalizes with the leukotriene (LT)-initiating enzyme 5-lipoxygenase (5-LO) in the MC nucleus. The NO donor, S-nitrosoglutathione (SNOG), inhibited, whereas the NOS inhibitor, N(G)-nitro-l-arginine methyl ester (L-NAME), potentiated LT release in a dose-dependent manner. Thus, human MC lines produce NO in both cytoplasmic and nuclear compartments, and endogenously produced NO can regulate LT production by MCs. PMID:15044250

  20. Redox-modulating agents target NOX2-dependent IKKε oncogenic kinase expression and proliferation in human breast cancer cell lines

    PubMed Central

    Mukawera, Espérance; Chartier, Stefany; Williams, Virginie; Pagano, Patrick J.; Lapointe, Réjean; Grandvaux, Nathalie

    2015-01-01

    Oxidative stress is considered a causative factor in carcinogenesis, but also in the development of resistance to current chemotherapies. The appropriate usage of redox-modulating compounds is limited by the lack of knowledge of their impact on specific molecular pathways. Increased levels of the IKKε kinase, as a result of gene amplification or aberrant expression, are observed in a substantial number of breast carcinomas. IKKε not only plays a key role in cell transformation and invasiveness, but also in the development of resistance to tamoxifen. Here, we studied the effect of in vitro treatment with the redox-modulating triphenylmethane dyes, Gentian Violet and Brilliant Green, and nitroxide Tempol on IKKε expression and cell proliferation in the human breast cancer epithelial cell lines exhibiting amplification of IKKε, MCF-7 and ZR75.1. We show that Gentian Violet, Brilliant Green and Tempol significantly decrease intracellular superoxide anion levels and inhibit IKKε expression and cell viability. Treatment with Gentian Violet and Brilliant Green was associated with a reduced cyclin D1 expression and activation of caspase 3 and/or 7. Tempol decreased cyclin D1 expression in both cell lines, while activation of caspase 7 was only observed in MCF-7 cells. Silencing of the superoxide-generating NOX2 NADPH oxidase expressed in breast cancer cells resulted in the significant reduction of IKKε expression. Taken together, our results suggest that redox-modulating compounds targeting NOX2 could present a particular therapeutic interest in combination therapy against breast carcinomas exhibiting IKKε amplification. PMID:26177467

  1. Transcriptome analysis of aldosterone-regulated genes in human vascular endothelial cell lines stably expressing mineralocorticoid receptor.

    PubMed

    Sekizawa, Naoko; Yoshimoto, Takanobu; Hayakawa, Eri; Suzuki, Noriko; Sugiyama, Toru; Hirata, Yukio

    2011-07-20

    A series of studies have demonstrated that endothelial cell is one of the target tissues of aldosterone. Here, we have conducted a transcriptome analysis of aldosterone-inducible genes in human endothelial cell lines stably expressing human mineralocorticoid receptor (MR) by retroviral system (MR-EAhy). We found that aldosterone in physiologic concentrations robustly induced MR-dependent transcriptional response in MR-EAhy. By DNA microarray analysis, we validated 12 aldosterone-up-regulated genes among which at least seven were concomitantly associated with increased protein expression. We also found five aldosterone-down-regulated genes. Among 11 aldosterone-up-regulated genes tested, mRNA expressions of three (ESM1, SNF1LK, ANGPTL4) were significantly up-regulated in aortic tissue from aldosterone-induced hypertensive rats compared to those from control rats, suggesting their potential pathophysiologic significance in vivo. In conclusion, using MR stably expressed human endothelial cell lines, we identified a variety of aldosterone-inducible genes, suggesting their possible roles in the development and/or the protection for aldosterone-induced vascular injury.

  2. Differential expression of melanoma-associated antigens and molecules involved in antigen processing and presentation in three cell lines established from a single patient.

    PubMed

    Kovalcsik, Edit; John, Justin; Turner, Matthew; Birchall, Lindsay; Sage, Deborah; Whittle, Robert; Dalgleish, Angus; Pandha, Hardev

    2004-12-01

    Tumour cells are able to evade the immune system by using several 'escape mechanisms'. Downregulation of molecules involved in the processing and presentation of self-antigens has been reported. However, these adaptations have not been compared in metastases in different anatomical locations but derived from a single patient. We investigated three melanoma cell lines--MJT1 from the parietal lobe of the brain, MJT3 from the cerebellum and MJT5 from the left side of the neck--established from biopsies excised from a 45 year old female patient. Although human leukocyte antigen (HLA) class I was detected in all three cell lines by flow cytometry using an anti-HLA monomorphic antibody, further serological analysis demonstrated HLA B38 loss in all three cell lines, HLA B7 downregulation in MJT5 (skin metastases) and B7 loss in MJT3 and MJT1 (brain metastases) compared with the HLA type of the patient's normal autologous lymphocytes. Interferon-gamma (IFNgamma) treatment increased the expression of HLA class I and transporters associated with antigen processing 1 (TAP1) in all three cell lines. De novo HLA class II molecule expression was observed after IFNgamma treatment in MJT3 and MJT5. Western blot and reverse transcription-polymerase chain reaction results revealed heterogeneity of melanoma-associated antigen (MAA) expression in the cell lines: MJT3 cells expressed higher levels of MAAs than the other two cell lines. In conclusion, this study has demonstrated that three metastatic lesions from a single patient can have differential expression of molecules involved in antigen processing (TAP1) and presentation (HLA I and II), but that expression of these molecules is modulated by IFNgamma to a similar degree in all cell lines. In contrast, the downregulation of expression of specific MAAs between the three cell lines was unaffected by the addition of IFNgamma.

  3. A cautionary note on cosmetics containing ingredients that increase aquaporin-3 expression.

    PubMed

    Verkman, A S

    2008-10-01

    Aquaporin-3 (AQP3) is a membrane transport protein that facilitates water and glycerol transport across cell plasma membranes in the basal layer of keratinocytes in normal skin. Motivated by a relation between AQP3 expression and skin water content, several companies have marketed cosmetics containing ingredients that increase AQP3 expression. However, caution seems warranted in targeting AQP3 to increase skin moisturization based on a recently discovered association in mice between epidermal AQP3 expression and skin tumor formation. PMID:18312385

  4. Increased range of ultrasonic guided wave testing of overhead transmission line cables using dispersion compensation.

    PubMed

    Legg, Mathew; Yücel, Mehmet K; Kappatos, Vassilios; Selcuk, Cem; Gan, Tat-Hean

    2015-09-01

    Overhead Transmission Line (OVTL) cables can experience structural defects and are, therefore, inspected using Non-Destructive Testing (NDT) techniques. Ultrasonic Guided Waves (UGW) is one NDT technique that has been investigated for inspection of these cables. For practical use, it is desirable to be able to inspect as long a section of cable as possible from a single location. This paper investigates increasing the UGW inspection range on Aluminium Conductor Steel Reinforced (ACSR) cables by compensating for dispersion using dispersion curve data. For ACSR cables, it was considered to be difficult to obtain accurate dispersion curves using modelling due to the complex geometry and unknown coupling between wire strands. Group velocity dispersion curves were, therefore, measured experimentally on an untensioned, 26.5m long cable and a method of calculating theoretical dispersion curves was obtained. Attenuation and dispersion compensation were then performed for a broadband Maximum Length Sequence (MLS) excitation signal. An increase in the Signal to Noise Ratio (SNR) of about 4-8dB compared to that of the dispersed signal was obtained. However, the main benefit was the increased ability to resolve the individual echoes from the end of the cable and an introduced defect in the form of a cut, which was 7 to at least 13dB greater than that of the dispersed signal. Five echoes were able to be clearly detected using MLS excitation signal, indicating the potential for an inspection range of up to 130m in each direction. To the best of the authors knowledge, this is the longest inspection range for ACSR cables reported in the literature, where typically cables, which were only one or two meter long, have been investigated previously. Narrow band tone burst and Hann windowed tone burst excitation signal also showed increased SNR and ability to resolve closely spaced echoes. PMID:25991388

  5. Chinese Hamster Ovary (CHO) Host Cell Engineering to Increase Sialylation of Recombinant Therapeutic Proteins by Modulating Sialyltransferase Expression

    PubMed Central

    Lin, Nan; Mascarenhas, Joaquina; Sealover, Natalie R.; George, Henry J.; Brooks, Jeanne; Kayser, Kevin J.; Gau, Brian; Yasa, Isil; Azadi, Parastoo; Archer-Hartmann, Stephanie

    2015-01-01

    N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio-better” protein therapeutics and cell culture vaccine production. PMID:25641927

  6. Histone deacetylase inhibitors promote the expression of ATP2A3 gene in breast cancer cell lines.

    PubMed

    Contreras-Leal, Erika; Hernández-Oliveras, Andrés; Flores-Peredo, Lucía; Zarain-Herzberg, Ángel; Santiago-García, Juan

    2016-10-01

    Recent studies have shown that expression of Sarco(endo)plasmic Reticulum Ca(2+) -ATPase 2 (SERCA2) is decreased in oral cancer; whereas expression of SERCA3 is considerably decreased or absent in human colon, gastric, breast, and lung cancers. The ATP2A2 and ATP2A3 genes encode SERCA2 and SERCA3 isoforms, respectively. Promoter methylation on CpG islands was responsible for the repression of ATP2A2 gene in human oral cancer samples. On the other hand, histone deacetylase inhibitors (HDACi) up-regulate ATP2A3 expression in gastric, colon, and lung cancer cells in culture, however, the molecular mechanism is unknown. In this study, we investigate whether HDACi and DNA methylation regulate ATP2A2 and ATP2A3 expression in human breast cancer cell lines. Results show a marked induction of SERCA3a and pan-SERCA3 mRNA expression in human MCF-7 and MDA-MB-231 cells treated with sodium butyrate (NaB) or trichostatin A (TSA); whereas SERCA2b mRNA expression did not change significantly. ChIP assays show that NaB or TSA treatment of MDA-MB-231 cells increases H3K9 acetylation on ATP2A3 promoter. NaB also decreases H3K9 trimethylation; suggesting that these modifications stimulate ATP2A3 gene expression, through a chromatin remodeling mechanism. In contrast, NaB or TSA do not increase H3K9-acetylation of ATP2A2 proximal promoter. In addition, treatment with 5-aza-2'-deoxycytidine did not affect SERCA2b and SERCA3a expression, suggesting that promoter methylation status does not alter their expression in these cell lines. We propose that alteration of SERCA2b/SERCA3a isoform expression ratio could affect calcium management within the cell, and thus, the cellular pathways regulated by calcium could be compromised, such as cellular proliferation or apoptosis. © 2015 Wiley Periodicals, Inc.

  7. Tick saliva regulates migration, phagocytosis, and gene expression in the macrophage-like cell line, IC-21.

    PubMed

    Kramer, Carolyn D; Poole, Nina M; Coons, Lewis B; Cole, Judith A

    2011-03-01

    We studied the effects of tick saliva on cell migration, cell signaling, phagocytosis, and gene expression in the murine macrophage cell line, IC-21. Saliva increased both basal- and platelet-derived growth factor (PDGF)-stimulated migration in IC-21 cells. However, saliva did not affect PDGF-stimulated extracellular signal-regulated kinase (ERK) activity. Zymosan-mediated interleukin-1 receptor associated kinase (IRAK) activity increased when cells were pretreated with saliva. Saliva suppressed phagocytosis of zymosan particles by IC-21 cells. An RT(2) Profiler™ PCR Array revealed that saliva regulates gene expression in a manner consistent with an immune response skewed toward a Th2 reaction, which is characterized by production of anti-inflammatory cytokines IL-4 and IL-10. Our results using IC-21 cells suggest that Dermacentor variabilis has evolved a mechanism for regulating macrophage function, which may contribute to the tick's ability to modulate immune function. PMID:21145320

  8. Differential expression of myc, max and RB1 genes in human gliomas and glioma cell lines.

    PubMed Central

    Hirvonen, H. E.; Salonen, R.; Sandberg, M. M.; Vuorio, E.; Västrik, I.; Kotilainen, E.; Kalimo, H.

    1994-01-01

    Deregulated expression of myc proto-oncogenes is implicated in several human neoplasias. We analysed the expression of c-myc, N-myc, L-myc, max and RB1 mRNAs in a panel of human gliomas and glioma cell lines and compared the findings with normal neural cells. The max and RB1 genes were included in the study because their protein products can interact with the Myc proteins, being thus putative modulators of Myc activity. Several gliomas contained c/L-myc mRNAs at levels higher than those in fetal brain, L-myc predominantly in grade II/III and c-myc in grade III gliomas. High-level N-myc expression was detected. In one small-cell glioblastoma and lower levels in five other gliomas. In contrast, glioma cell lines totally lacked N/L-myc expression. The in situ hybridisations revealed mutually exclusive topographic distribution of myc and glial fibrillary acidic protein (GFAP) mRNAs, and a lack of correlation between myc expression and proliferative activity, max and RB1 mRNAs were detected in most tumours and cell lines. The glioma cells displayed interesting alternative splicing patterns of max mRNAs encoding Max proteins which either suppress (Max) or augment (delta Max) the transforming activity of Myc. We conclude that (1) glioma cells in vivo may coexpress several myc genes, thus resembling fetal neural cells; but (2) cultured glioma cells expression only c-myc; (3) myc, max and RB1 are regulated independently in glioma cells; and (4) alternative processing of max mRNA in some glioma cells results in delta Max encoding mRNAs not seen in normal fetal brain. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8286200

  9. Generation and gene expression profiling of 48 transcription-factor-inducible mouse embryonic stem cell lines

    PubMed Central

    Yamamizu, Kohei; Sharov, Alexei A.; Piao, Yulan; Amano, Misa; Yu, Hong; Nishiyama, Akira; Dudekula, Dawood B.; Schlessinger, David; Ko, Minoru S. H.

    2016-01-01

    Mouse embryonic stem cells (ESCs) can differentiate into a wide range – and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously, we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this “NIA Mouse ESC Bank,” we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs. PMID:27150017

  10. miRNAs expressed differently in cancer stem cells and cancer cells of human gastric cancer cell line MKN-45.

    PubMed

    Golestaneh, Azadeh Fahim; Atashi, Amir; Langroudi, Lida; Shafiee, Abbas; Ghaemi, Nasser; Soleimani, Masoud

    2012-07-01

    Recent studies show that cancers may originate from special cells named cancer stem cells (CSCs). As miRNAs have a prominent role in regulating cell activities, a question arise, that is, if there is any difference in miRNA expression level between CSC and other cancer cells of human gastric cancer cell line MKN-45. In this study, CSCs were isolated by fluorescence-activated cell sorter based on the expression level of cell surface marker CD44. CSC characteristics were checked using spheroid formation assay and soft agar assay. Using reverse transcriptase polymerase chain reaction (RT-PCR), the expression level of some stemness genes was studied. Real-time q-PCR was used for analysis of the expression level of miRNAs. CSCs were able to make spheroids and colonies, whereas other cancer cells failed to show aforementioned features. In addition, RT-PCR resulted in a difference in the expression levels of Nanog, Sox2, Lin28 and Oct-4 between these two kinds of cells. Real-time RT-PCR analysis demonstrated an increase in mir-21 and mir-302 expression level in CSCs, relative to cancer cells, whereas let-7a expression level was decreased in CSC in comparison with cancer cells, which may be due to their different differentiation level. On the other hand, mir-372, mir-373 and mir-520c-5p were markedly increased in cancer cells in comparison with CSCs. This study shows that there is a difference in miRNA expression level between CSCs and other cancer cells, which reflects dissimilar molecular pathways in these cells. These miRNAs may be promising objects for targeting CSCs specifically and efficiently.

  11. Ectopic expression of SlAGO7 alters leaf pattern and inflorescence architecture and increases fruit yield in tomato.

    PubMed

    Lin, Dongbo; Xiang, Ya; Xian, Zhiqiang; Li, Zhengguo

    2016-08-01

    ARGONAUTE7 (AGO7), a key regulator of the trans-acting small interfering RNAs (ta-siRNA) pathway, plays a conserved role in controlling leaf pattern among species. However, little is known about the ta-siRNA pathway in regulating inflorescence architecture and fruit yield. In this study, we characterized the expression pattern, subcellular localization and developmental functions of SlAGO7 in tomato (Solanum lycopersicum). Overexpressing SlAGO7 in tomato exhibited pleiotropic phenotypes, including improved axillary bud formation, altered leaf morphology and inflorescence architecture, and increased fruit yield. Cross-sectioning of leaves showed that the number of vascular bundles was significantly increased in 35:SlAGO7 lines. Overexpression of SlAGO7 increased the production of ta-siRNA, and repressed the expression ta-siRNA-targeted genes (SlARF2a, SlARF2b, SlARF3 and SlARF4). Further analysis showed that overexpression of SlAGO7 alters the expression of key genes implicated in leaf morphology, inflorescence architecture, auxin transport and signaling. In addition, the altered auxin response of 35:SlAGO7 lines were also investigated. These results suggested that SlAGO7 plays a positive role in determining inflorescence architecture and fruit yield though the ta-siRNA pathway. Therefore, SlAGO7 represents a useful gene that can be incorporated in tomato breeding programs for developing cultivars with yield potential. PMID:26847714

  12. Ectopic expression of SlAGO7 alters leaf pattern and inflorescence architecture and increases fruit yield in tomato.

    PubMed

    Lin, Dongbo; Xiang, Ya; Xian, Zhiqiang; Li, Zhengguo

    2016-08-01

    ARGONAUTE7 (AGO7), a key regulator of the trans-acting small interfering RNAs (ta-siRNA) pathway, plays a conserved role in controlling leaf pattern among species. However, little is known about the ta-siRNA pathway in regulating inflorescence architecture and fruit yield. In this study, we characterized the expression pattern, subcellular localization and developmental functions of SlAGO7 in tomato (Solanum lycopersicum). Overexpressing SlAGO7 in tomato exhibited pleiotropic phenotypes, including improved axillary bud formation, altered leaf morphology and inflorescence architecture, and increased fruit yield. Cross-sectioning of leaves showed that the number of vascular bundles was significantly increased in 35:SlAGO7 lines. Overexpression of SlAGO7 increased the production of ta-siRNA, and repressed the expression ta-siRNA-targeted genes (SlARF2a, SlARF2b, SlARF3 and SlARF4). Further analysis showed that overexpression of SlAGO7 alters the expression of key genes implicated in leaf morphology, inflorescence architecture, auxin transport and signaling. In addition, the altered auxin response of 35:SlAGO7 lines were also investigated. These results suggested that SlAGO7 plays a positive role in determining inflorescence architecture and fruit yield though the ta-siRNA pathway. Therefore, SlAGO7 represents a useful gene that can be incorporated in tomato breeding programs for developing cultivars with yield potential.

  13. Placental alkaline phosphatase isoenzyme expression by the non-HeLa DoT cervical-carcinoma cell line.

    PubMed Central

    Kottel, R H; Fishman, W H

    1981-01-01

    Expression of the oncodevelopmental protein, placental alkaline phosphatase, was observed in DoT cells, an epidermoid cell line derived from cervical carcinoma. Under normal conditions of growth in vitro, biochemical inhibition, cytochemical and immunological studies revealed that these cells express the term-placental (Regan) isoenzyme. Thus alkaline phosphatase activity was observed to be heat-stable and inhibited by L-phenylalanine. These properties, supported by immunoelectrophoretic analysis using antisera specific for liver, intestinal or term-placental isoenzymes, identified the isoenzyme as placental type. DoT cells treated with prednisolone (1 microgram/ml) increased total alkaline phosphatase specific activity. This activity was also identified as term-placental phosphatase isoenzyme. On the other hand, treatment of the same cells with sodium butyrate (1 mM) did not induce increased activity of the term-placental isoenzyme, an unexpected observation. As a result of these studies, DoT cells are proposed as a representative cell line for studies of the regulation of oncodevelopmental gene expression in human tumour cells of cervical origin. Images Fig. 2. Fig. 3. PMID:7342975

  14. Epigallocatechin-3-gallate Increases RXRγ-mediated Pro-apoptotic and Anti-invasive Effects in Gastrointestinal Cancer Cell Lines.

    PubMed

    Papi, Alessio; Govoni, Marzia; Ciavarella, Carmen; Spisni, Enzo; Orlandi, Marina; Farabegoli, Fulvia

    2016-01-01

    Molecules with synergistic effects often enhance the benefits of cancer therapy. We observed that the major catechin of green tea, (-)-Epigallocatechin-3-gallate (EGCG), induced retinoid X receptor-γ (RXRγ) expression in the SK-Ch-A1 cholangiocarcinoma cell line and in two colon carcinoma cell lines (LoVo and the derivative multi-drug resistant LoVoMDR). On this basis, we analyzed the effects of EGCG in combination with an RXRγ ligand, 6-OH-11-O-hydroxyphenantrene (IIF), or with a ligand of retinoic acid receptor, all-trans-retinoic acid (RA). IIF alone and in combination with EGCG activated the retinoic X response elements and induced the germ cell nuclear factor. In parallel, EGCG induced 67 kDa laminin receptor expression alone and in combination with IIF. We observed a synergistic growth inhibition with EGCG and IIF in combination at lower doses. These effects were accompanied by apoptosis activation through the mitochondrial pathway. Moreover, in LoVo cell line we observed an induction of Forkhead box O3 expression, another molecule involved in apoptosis activation. Finally, metalloproteinase activity and extracellular matrix metalloproteinase inducer (EMMPRIN) expression were inhibited and tumor cell invasion was strongly reduced in the SK-Ch-A1 cell line after treatment with EGCG and IIF. In conclusion, the use of specific RXR ligands in combination with catechins could open a new perspective in gastrointestinal tumor chemoprevention. PMID:26278714

  15. Conditional Expression of the Androgen Receptor Increases Susceptibility of Bladder Cancer in Mice

    PubMed Central

    Luong, Richard; Yu, Eun-Jeong; He, Yongfeng; Gonzalgo, Mark L.; Sun, Zijie

    2016-01-01

    Bladder cancer represents a significant human tumor burden, accounting for about 7.7% and 2.4% of all cancer cases in males and females, respectively. While men have a higher risk of developing bladder cancer, women tend to present at a later stage of disease and with more aggressive tumors. Previous studies have suggested a promotional role of androgen signaling in enhancing bladder cancer development. To directly assess the role of androgens in bladder tumorigenesis, we have developed a novel transgenic mouse strain, R26hARLoxP/+:Upk3aGCE/+, in which the human AR transgene is conditionally expressed in bladder urothelium. Intriguingly, both male and female R26hARLoxP/+:Upk3aGCE/+ mice display a higher incidence of urothelial cell carcinoma (UCC) than the age and sex matched control littermates in response to the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). We detect expression of the human AR transgene in CK5-positive and p63-positive basal cells in bladder urothelium. Further analyses of UCC tissues from R26hARLoxP/+:Upk3aGCE/+ mice showed that the majority of tumor cells are of urothelial basal cell origin. Positive immunostaining of transgenic AR protein was observed in the majority of tumor cells of the transgenic mice, providing a link between transgenic AR expression and oncogenic transformation. We observed an increase in Ki67 positive cells within the UCC lesions of transgenic AR mice. Manipulating endogenous androgen levels by castration and androgen supplementation directly affected bladder tumor development in male and female R26hARLoxP/+:Upk3aGCE/+ mice, respectively. Taken together, our data demonstrate for the first time that conditional activation of transgenic AR expression in bladder urothelium enhances carciongen-induced bladder tumor formation in mice. This new AR transgenic mouse line mimics certain features of human bladder cancer and can be used to study bladder tumorigenesis and for drug development. PMID:26862755

  16. Expression of LINE-1 retroposons is essential for murine preimplantation development.

    PubMed

    Beraldi, Rosanna; Pittoggi, Carmine; Sciamanna, Ilaria; Mattei, Elisabetta; Spadafora, Corrado

    2006-03-01

    In higher eukaryotes, reverse transcriptase (RT) activities are encoded by a variety of endogenous retroviruses and retrotransposable elements. We previously found that mouse preimplantation embryos are endowed with an endogenous RT activity. Inhibition of that activity by the non nucleosidic inhibitor nevirapine or by microinjection of anti-RT antibody caused early embryonic developmental arrest. Those experiments indicated that RT is required for early development, but did not identify the responsible coding elements. We now show that microinjection of morpholino-modified antisense oligonucleotides targeting the 5' end region of active LINE-1 retrotransposons in murine zygotes irreversibly arrests preimplantation development at the two- and four-cell stages; the overall level of functional RT is concomitantly downregulated in arrested embryos. Furthermore, we show that the induction of embryo developmental arrest is associated with a substantial reprogramming of gene expression. Together, these results support the conclusion that expression of LINE-1 retrotransposons is required for early embryo preimplantation development.

  17. Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

    SciTech Connect

    Takahashi, Nobuhiko; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. Black-Right-Pointing-Pointer Adipose lipin-1 expression is reduced in obesity. Black-Right-Pointing-Pointer Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. Black-Right-Pointing-Pointer Lipin-1 is involved in adipose inflammation. -- Abstract: Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-{kappa}B activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

  18. Loss of N-Cadherin Expression in Tumor Transplants Produced From As+3- and Cd+2-Transformed Human Urothelial (UROtsa) Cell Lines

    PubMed Central

    Sandquist, Elizabeth J.; Somji, Seema; Dunlevy, Jane R.; Garrett, Scott H.; Zhou, Xu Dong; Slusser-Nore, Andrea

    2016-01-01

    Background Epithelial to mesenchymal transition is a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, epithelial to mesenchymal transition has been proposed to play an important role during specific stages of tumor progression. The role epithelial to mesenchymal transition and mesenchymal to epithelial transition might play in toxicant-induced urothelial cancer is unknown. Methods Real-time PCR, Western blotting, immuno-histochemistry and immuno-fluorescence were used to determine the expression of E- and N-cadherin in the UROtsa parent, the As+3- and Cd+2-transformed cell lines, the spheroids isolated from these cell lines as well as the tumor heterotransplants that were produced by the injection of the transformed cells into immune compromised mice. Results This study showed that N-cadherin expression was increased in 6 As+3- and 7 Cd+2- transformed cell lines generated from human urothelial cells (UROtsa). The expression varied within each cell line, with 10% to 95% of the cells expressing N-cadherin. Tumors produced from these cell lines showed no expression of the N-cadherin protein. Spheroids which are made up of putative cancer initiating cells produced from these cell lines showed only background expression of N-cadherin mRNA, increased expression of aldehyde dehydrogenase 1 mRNA and produced tumors which did not express N-cadherin. There was no change in the expression of E-cadherin in the tumors, and the tumors formed by all the As+3 and Cd+2-transformed cell lines and cancer initiating cells stained intensely and uniformly for E-cadherin. Conclusions The finding that the cells expressing N-cadherin gave rise to tumors with no expression of N-cadherin is in agreement with the classical view of epithelial to mesenchymal transition. Epithelial to mesenchymal transition and N-cadherin are associated with dissemination and not with the ability to establish new tumor growth

  19. Expression and precursor processing of neuropeptide Y in human and murine neuroblastoma and pheochromocytoma cell lines.

    PubMed

    O'Hare, M M; Schwartz, T W

    1989-12-15

    The synthesis and processing of the precursor for neuropeptide Y (NPY) were studied in 16 human and murine neuroendocrine cell lines. Eight of the cell lines, NS-20Y, PC12, LA-N-5, CHP-234, SMS-KCNR, SH-SY5Y, SMS-KCN, and BE(2)-M17, produced sufficient quantities to permit chromatographic characterization of the NPY immunoreactivity. Although the cell lines varied in the amount of NPY they produced, both within and between cell lines, they displayed a relatively constant pattern of posttranslational modifications. In contrast to observations in tumor extracts (M. M. T. O'Hare and T. W. Schwartz, Cancer Res., 49: 7010-7014, 1989), all cell lines studied contained a substantial amount of the intracellular NPY in the form of the unprocessed propeptide, 57% (range, 33-72%) as characterized by both gel filtrations (32 experiments in 8 cell lines) and "in vitro conversion" with endoproteinase Lys-C. In the majority, 4 of 6 cell lines studied, almost all of the NPY, which by size corresponded to the mature 36-amino acid form, was amidated as assessed by isoelectric focusing and by a radioimmunoassay specific for the COOH-terminal amide group of the peptide. Both the propeptide and smaller molecular forms of NPY were secreted from the cell cultures; however, proteolytic degradation in the tissue culture medium prevented a detailed, meaningful characterization of these peptides. It is concluded that many neuroendocrine cell lines, especially those derived from human neuroblastomas, express the NPY gene; the cells display a partly impaired dibasic processing capacity but they generally amidate the products efficiently.

  20. Effect of Thymoquinone on P53 Gene Expression and Consequence Apoptosis in Breast Cancer Cell Line

    PubMed Central

    Dastjerdi, Mehdi Nikbakht; Mehdiabady, Ebrahim Momeni; Iranpour, Farhad Golshan; Bahramian, Hamid

    2016-01-01

    Background: Nigella sativa has been a nutritional flavoring factor and natural treatment for many ailments for so many years in medical science. Earlier studies have been reported that thymoquinone (TQ), an active compound of its seed, contains anticancer properties. Previous studies have shown that TQ induces apoptosis in breast cancer cells but it is unclear the role of P53 in the apoptotic pathway. Hereby, this study reports the potency of TQ on expression of tumor suppressor gene P53 and apoptosis induction in breast cancer cell line Michigan Cancer Foundation-7 (MCF-7). Methods: MCF-7 cell line was cultured and treated with TQ, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out for evaluating the half-maximal inhibitory concentration (IC50) values after 24 h of treatment. The percentage of apoptotic cells was measured by flow cytometry. Real-time polymerase chain reaction (PCR) was performed to estimate the messenger RNA expression of P53 in MCF-7 cell line at different times. Results: The IC50 value for the TQ in MCF-7 cells was 25 μM that determined using MTT assay. The flow cytometry and real-time PCR results showed that TQ could induce apoptosis in MCF-7 cells, and the P53 gene expression was dramatically up-regulated by ascending time, respectively. Hence, there was significant difference in 48 and 72 h. Conclusions: Our results demonstrated that TQ could induce apoptosis in MCF-7 cells through up-regulation of P53 expression in breast cancer cell line (MCF-7) by time-dependent manner. PMID:27141285

  1. An Empirical Expression for Line Widths of Ammonia from Far-Infrared Measurements

    NASA Technical Reports Server (NTRS)

    Brown, L.; Peterson, D.

    1994-01-01

    The hydrogen- and self-broadened line widths of 116 (sup 14)NH(sub 3) ground state transitions with J,K = 1,0 to 10,10 have been measured at 0.0006 cm(sup -1) resolution using a Bruker spectrometer between 40 to 210 cm(sup -1). These experimental widths have been reproduced to 2.4% and 11% respectively using an heuristically derived expression of the form....

  2. A silencer inhibitor confers specific expression of intestinal trefoil factor in gobletlike cell lines.

    PubMed

    Iwakiri, D; Podolsky, D K

    2001-06-01

    Intestinal trefoil factor (ITF) is selectively expressed in intestinal goblet cells. Previous studies identified cis-regulatory elements in the proximal promoter of ITF, but these were insufficient to recapitulate the exquisite tissue- and cell-specific expression of native ITF in vivo. Preliminary studies suggested that goblet cell-specific expression of murine ITF requires elements far upstream that include a silencer element that effectively prevents ITF expression in non-goblet cells. Transient transfection studies using native or mutant ITF 5'-flanking sequences identified a region that restores expression in goblet cells. This element, designated goblet cell silencer inhibitor (GCSI) element, enables human and murine goblet cell-like cell lines to override the silencing effect of more proximal elements. The GCSI has no intrinsic enhancer activity and regulates expression only when the silencer element is present. Ligation of GCSI and silencer elements to sucrase-isomaltase conferred goblet cell-specific expression. Goblet cells but not non-goblet cells possess a nuclear protein that binds to the GCSI regulatory element (GCSI binding protein; GCSI-BP). Both transient transfection and gel mobility shift assay studies localize the GCSI and GCSI-BP to -2216 to -2204. We conclude that goblet cell-specific transcription of ITF in vivo depends on a regulatory element designated GCSI.

  3. Identification of a Ly-6 superfamily gene expressed in lateral line neuromasts in zebrafish.

    PubMed

    Ji, Dongrui; Li, Lingyi; Zhang, Shicui; Li, Hongyan

    2015-01-01

    Lymphocyte antigen-6 (Ly-6) superfamily members have been identified in zebrafish, but the expression and function of these Ly-6 genes remain largely unknown. Posterior lateral line (pLL) system is produced by migrating pLL primordium (pLLp). Chemokine signaling, Notch, Wnt, and fibroblast growth factor (FGF) signaling regulate migration of pLLp cells and formation of neuromasts. However, the mechanism of neuromast deposition remains to be explored. Identification of novel genes expressed in pLLp will certainly help the study of such a process. Here we identified a Ly-6 gene called neuromast-expressed gpi-anchored lymphocyte antigen-6 (negaly6), which was specifically expressed in neuromast. Quantitative real-time PCR (qRT-PCR) analysis showed that negaly6 started to be expressed at 24 hpf, and whole-mount in situ hybridization analysis indicated that negaly6 was highly expressed in the trailing zone of pLLp and mature neuromast. Furthermore, negaly6 expression was inhibited by FGF signaling antagonist but not by Wnt signaling agonist or antagonist. Collectively, these data indicate that negaly6 may be associated with the regulation of neuromast deposition via FGF signaling pathway.

  4. Murine bone cell lines as models for spaceflight induced effects on differentiation and gene expression

    NASA Astrophysics Data System (ADS)

    Lau, P.; Hellweg, C. E.; Baumstark-Khan, C.; Reitz, G.

    Critical health factors for space crews especially on long-term missions are radiation exposure and the absence of gravity DNA double strand breaks DSB are presumed to be the most deleterious DNA lesions after radiation as they disrupt both DNA strands in close proximity Besides radiation risk the absence of gravity influences the complex skeletal apparatus concerning muscle and especially bone remodelling which results from mechanical forces exerting on the body Bone is a dynamic tissue which is life-long remodelled by cells from the osteoblast and osteoclast lineage Any imbalance of this system leads to pathological conditions such as osteoporosis or osteopetrosis Osteoblastic cells play a crucial role in bone matrix synthesis and differentiate either into bone-lining cells or into osteocytes Premature terminal differentiation has been reported to be induced by a number of DNA damaging or cell stress inducing agents including ionising and ultraviolet radiation as well as treatment with mitomycin C In the present study we compare the effects of sequential differentiation by adding osteoinductive substances ss -glycerophosphate and ascorbic acid Radiation-induced premature differentiation was investigated regarding the biosynthesis of specific osteogenic marker molecules and the differentiation dependent expression of marker genes The bone cell model established in our laboratory consists of the osteocyte cell line MLO-Y4 the osteoblast cell line OCT-1 and the subclones 4 and 24 of the osteoblast cell line MC3T3-E1 expressing several

  5. Ectopic phytocystatin expression increases nodule numbers and influences the responses of soybean (Glycine max) to nitrogen deficiency.

    PubMed

    Quain, Marian D; Makgopa, Matome E; Cooper, James W; Kunert, Karl J; Foyer, Christine H

    2015-04-01

    Cysteine proteases and cystatins have many functions that remain poorly characterised, particularly in crop plants. We therefore investigated the responses of these proteins to nitrogen deficiency in wild-type soybeans and in two independent transgenic soybean lines (OCI-1 and OCI-2) that express the rice cystatin, oryzacystatin-I (OCI). Plants were grown for four weeks under either a high (5 mM) nitrate (HN) regime or in the absence of added nitrate (LN) in the absence or presence of symbiotic rhizobial bacteria. Under the LN regime all lines showed similar classic symptoms of nitrogen deficiency including lower shoot biomass and leaf chlorophyll. However, the LN-induced decreases in leaf protein and increases in root protein tended to be smaller in the OCI-1 and OCI-2 lines than in the wild type. When LN-plants were grown with rhizobia, OCI-1 and OCI-2 roots had significantly more crown nodules than wild-type plants. The growth nitrogen regime had a significant effect on the abundance of transcripts encoding vacuolar processing enzymes (VPEs), LN-dependent increases in VPE2 and VPE3 transcripts in all lines. However, the LN-dependent increases of VPE2 and VPE3 transcripts were significantly lower in the leaves of OCI-1 and OCI-2 plants than in the wild type. These results show that nitrogen availability regulates the leaf and root cysteine protease, VPE and cystatin transcript profiles in a manner that is in some cases influenced by ectopic OCI expression. Moreover, the OCI-dependent inhibition of papain-like cysteine proteases favours increased nodulation and enhanced tolerance to nitrogen limitation, as shown by the smaller LN-dependent decreases in leaf protein observed in the OCI-1 and OCI-2 plants relative to the wild type.

  6. Identification of circadian-related gene expression profiles in entrained breast cancer cell lines.

    PubMed

    Gutiérrez-Monreal, Miguel A; Treviño, Victor; Moreno-Cuevas, Jorge E; Scott, Sean-Patrick

    2016-01-01

    Cancer cells have broken circadian clocks when compared to their normal tissue counterparts. Moreover, it has been shown in breast cancer that disruption of common circadian oscillations is associated with a more negative prognosis. Numerous studies, focused on canonical circadian genes in breast cancer cell lines, have suggested that there are no mRNA circadian-like oscillations. Nevertheless, cancer cell lines have not been extensively characterized and it is unknown to what extent the circadian oscillations are disrupted. We have chosen representative non-cancerous and cancerous breast cell lines (MCF-10A, MCF-7, ZR-75-30, MDA-MB-231 and HCC-1954) in order to determine the degree to which the circadian clock is damaged. We used serum shock to synchronize the circadian clocks in culture. Our aim was to initially observe the time course of gene expression using cDNA microarrays in the non-cancerous MCF-10A and the cancerous MCF-7 cells for screening and then to characterize specific genes in other cell lines. We used a cosine function to select highly correlated profiles. Some of the identified genes were validated by quantitative polymerase chain reaction (qPCR) and further evaluated in the other breast cancer cell lines. Interestingly, we observed that breast cancer and non-cancerous cultured cells are able to generate specific circadian expression profiles in response to the serum shock. The rhythmic genes, suggested via microarray and measured in each particular subtype, suggest that each breast cancer cell type responds differently to the circadian synchronization. Future results could identify circadian-like genes that are altered in breast cancer and non-cancerous cells, which can be used to propose novel treatments. Breast cell lines are potential models for in vitro studies of circadian clocks and clock-controlled pathways. PMID:27010605

  7. Increasing nitric oxide content in Arabidopsis thaliana by expressing rat neuronal nitric oxide synthase resulted in enhanced stress tolerance.

    PubMed

    Shi, Hai-Tao; Li, Rong-Jun; Cai, Wei; Liu, Wen; Wang, Chao-Lun; Lu, Ying-Tang

    2012-02-01

    Nitric oxide (NO) plays essential roles in many physiological and developmental processes in plants, including biotic and abiotic stresses, which have adverse effects on agricultural production. However, due to the lack of findings regarding nitric oxide synthase (NOS), many difficulties arise in investigating the physiological roles of NO in vivo and thus its utilization for genetic engineering. Here, to explore the possibility of manipulating the endogenous NO level, rat neuronal NOS (nNOS) was expressed in Arabidopsis thaliana. The 35S::nNOS plants showed higher NOS activity and accumulation of NO using the fluorescent probe 3-amino, 4-aminomethyl-2', 7'-difluorescein, diacetate (DAF-FM DA) assay and the hemoglobin assay. Compared with the wild type, the 35S::nNOS plants displayed improved salt and drought tolerance, which was further confirmed by changes in physiological parameters including reduced water loss rate, reduced stomatal aperture, and altered proline and malondialdehyde content. Quantitative real-time PCR analyses revealed that the expression of several stress-regulated genes was up-regulated in the transgenic lines. Furthermore, the transgenic lines also showed enhanced disease resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 by activating the expression of defense-related genes. In addition, we found that the 35S::nNOS lines flowered late by regulating the expression of CO, FLC and LFY genes. Together, these results demonstrated that it is a useful strategy to exploit the roles of plant NO in various processes by the expression of rat nNOS. The approach may also be useful for genetic engineering of crops with increased environmental adaptations. PMID:22186181

  8. Reactive oxygen species production and antioxidant enzyme expression after Epstein-Barr virus lytic cycle induction in Raji cell line.

    PubMed

    Gargouri, Bochra; Nasr, Rihab; ben Mansour, Riadh; Lassoued, Saloua; Mseddi, Malek; Attia, Hammadi; El Feki, Abd el Fatteh; Van Pelt, Jos

    2011-12-01

    In a previous study, we have described oxidative stress during Epstein-Barr virus lytic cycle induction. Oxidative stress was evidenced by the observed high MDA levels and the decreased activities of antioxidant enzymes. We hypothesised that the lower activities of the antioxidant enzymes decrease were the result of either the excessive production of reactive oxygen radical species (ROS) or a negative regulation of the antioxidant enzyme gene expressions. In an attempt to clarify this situation, EBV lytic cycle was induced in Raji cell line by a non-stressing dose of 12-0-tetradecanoylphorbol-13-acetate. BZLF-1, superoxide dismutase, and catalase gene expressions were then analysed using semi-quantitative RT-PCR, simultaneously at a kinetic of 6, 12, 24, 36, and 48 h. ROS production was evaluated by chemiluminescence. A study was conducted to establish whether ROS production, BZLF-1, and the expression of antioxidant genes were inter-correlated. Induction of the lytic cycle resulted in increased expressions of the genes of superoxide dismutase and catalase, which began at 24 h (p < 0.05) and reached a peak at 48 h (p < 0.05). Significant increases of the ROS levels were observed in TPA-treated Raji cell line at 12 h, as compared with untreated cells, reaching a peak at 48 h after EBV lytic cycle induction. ROS production correlates positively with BZLF-1, SOD, and CAT gene expressions (p < 0.05; r = 0.913, r = 0.978, and r = 0.955, respectively). A positive correlation was also observed between BZLF-1 and antioxidant gene expressions (p < 0.05; r = 0.961 and r = 0.987, respectively). In conclusion, the observed increases of the SOD and CAT gene expressions eliminate the hypothesis of a repression of the respective genes during the induction of the lytic cycle. On the other hand, the observed direct correlation between the BZLF-1 gene expression and the ROS production is indicative of a role of this gene in oxidative stress.

  9. A Minimalistic Resource Allocation Model to Explain Ubiquitous Increase in Protein Expression with Growth Rate

    PubMed Central

    Keren, Leeat; Segal, Eran; Milo, Ron

    2016-01-01

    Most proteins show changes in level across growth conditions. Many of these changes seem to be coordinated with the specific growth rate rather than the growth environment or the protein function. Although cellular growth rates, gene expression levels and gene regulation have been at the center of biological research for decades, there are only a few models giving a base line prediction of the dependence of the proteome fraction occupied by a gene with the specific growth rate. We present a simple model that predicts a widely coordinated increase in the fraction of many proteins out of the proteome, proportionally with the growth rate. The model reveals how passive redistribution of resources, due to active regulation of only a few proteins, can have proteome wide effects that are quantitatively predictable. Our model provides a potential explanation for why and how such a coordinated response of a large fraction of the proteome to the specific growth rate arises under different environmental conditions. The simplicity of our model can also be useful by serving as a baseline null hypothesis in the search for active regulation. We exemplify the usage of the model by analyzing the relationship between growth rate and proteome composition for the model microorganism E.coli as reflected in recent proteomics data sets spanning various growth conditions. We find that the fraction out of the proteome of a large number of proteins, and from different cellular processes, increases proportionally with the growth rate. Notably, ribosomal proteins, which have been previously reported to increase in fraction with growth rate, are only a small part of this group of proteins. We suggest that, although the fractions of many proteins change with the growth rate, such changes may be partially driven by a global effect, not necessarily requiring specific cellular control mechanisms. PMID:27073913

  10. Oxytocin increases bias, but not accuracy, in face recognition line-ups.

    PubMed

    Bate, Sarah; Bennetts, Rachel; Parris, Benjamin A; Bindemann, Markus; Udale, Robert; Bussunt, Amanda

    2015-07-01

    Previous work indicates that intranasal inhalation of oxytocin improves face recognition skills, raising the possibility that it may be used in security settings. However, it is unclear whether oxytocin directly acts upon the core face-processing system itself or indirectly improves face recognition via affective or social salience mechanisms. In a double-blind procedure, 60 participants received either an oxytocin or placebo nasal spray before completing the One-in-Ten task-a standardized test of unfamiliar face recognition containing target-present and target-absent line-ups. Participants in the oxytocin condition outperformed those in the placebo condition on target-present trials, yet were more likely to make false-positive errors on target-absent trials. Signal detection analyses indicated that oxytocin induced a more liberal response bias, rather than increasing accuracy per se. These findings support a social salience account of the effects of oxytocin on face recognition and indicate that oxytocin may impede face recognition in certain scenarios. PMID:25433464

  11. Oxytocin increases bias, but not accuracy, in face recognition line-ups.

    PubMed

    Bate, Sarah; Bennetts, Rachel; Parris, Benjamin A; Bindemann, Markus; Udale, Robert; Bussunt, Amanda

    2015-07-01

    Previous work indicates that intranasal inhalation of oxytocin improves face recognition skills, raising the possibility that it may be used in security settings. However, it is unclear whether oxytocin directly acts upon the core face-processing system itself or indirectly improves face recognition via affective or social salience mechanisms. In a double-blind procedure, 60 participants received either an oxytocin or placebo nasal spray before completing the One-in-Ten task-a standardized test of unfamiliar face recognition containing target-present and target-absent line-ups. Participants in the oxytocin condition outperformed those in the placebo condition on target-present trials, yet were more likely to make false-positive errors on target-absent trials. Signal detection analyses indicated that oxytocin induced a more liberal response bias, rather than increasing accuracy per se. These findings support a social salience account of the effects of oxytocin on face recognition and indicate that oxytocin may impede face recognition in certain scenarios.

  12. Simvastatin induces mitochondrial dysfunction and increased atrogin-1 expression in H9c2 cardiomyocytes and mice in vivo.

    PubMed

    Bonifacio, Annalisa; Mullen, Peter J; Mityko, Ileana Scurtu; Navegantes, Luiz C; Bouitbir, Jamal; Krähenbühl, Stephan

    2016-01-01

    Simvastatin is effective and well tolerated, with adverse reactions mainly affecting skeletal muscle. Important mechanisms for skeletal muscle toxicity include mitochondrial impairment and increased expression of atrogin-1. The aim was to study the mechanisms of toxicity of simvastatin on H9c2 cells (a rodent cardiomyocyte cell line) and on the heart of male C57BL/6 mice. After, exposure to 10 μmol/L simvastatin for 24 h, H9c2 cells showed impaired oxygen consumption, a reduction in the mitochondrial membrane potential and a decreased activity of several enzyme complexes of the mitochondrial electron transport chain (ETC). The cellular ATP level was also decreased, which was associated with phosphorylation of AMPK, dephosphorylation and nuclear translocation of FoxO3a as well as increased mRNA expression of atrogin-1. Markers of apoptosis were increased in simvastatin-treated H9c2 cells. Treatment of mice with 5 mg/kg/day simvastatin for 21 days was associated with a 5 % drop in heart weight as well as impaired activity of several enzyme complexes of the ETC and increased mRNA expression of atrogin-1 and of markers of apoptosis in cardiac tissue. Cardiomyocytes exposed to simvastatin in vitro or in vivo sustain mitochondrial damage, which causes AMPK activation, dephosphorylation and nuclear transformation of FoxO3a as well as increased expression of atrogin-1. Mitochondrial damage and increased atrogin-1 expression are associated with apoptosis and increased protein breakdown, which may cause myocardial atrophy.

  13. Neuropilin2 expressed in gastric cancer endothelial cells increases the proliferation and migration of endothelial cells in response to VEGF

    SciTech Connect

    Kim, Woo Ho; Lee, Sun Hee; Jung, Myung Hwan; Seo, Ji Heun; Kim, Jin; Kim, Min A; Lee, You Mie

    2009-08-01

    The structure and characteristics of the tumor vasculature are known to be different from those of normal vessels. Neuropilin2 (Nrp2), which is expressed in non-endothelial cell types, such as neuronal or cancer cells, functions as a receptor for both semaphorin and vascular endothelial growth factor (VEGF). After isolating tumor and normal endothelial cells from advanced gastric cancer tissue and normal gastric mucosa tissues, respectively, we identified genes that were differentially expressed in gastric tumor endothelial (TEC) and normal endothelial cells (NEC) using DNA oligomer chips. Using reverse transcriptase-PCR, we confirmed the chip results by showing that Nrp2 gene expression is significantly up-regulated in TEC. Genes that were found to be up-regulated in TEC were also observed to be up-regulated in human umbilical vein endothelial cells (HUVECs) that were co-cultured with gastric cancer cells. In addition, HUVECs co-cultured with gastric cancer cells showed an increased reactivity to VEGF-induced proliferation and migration. Moreover, overexpression of Nrp2 in HUVECs significantly enhanced the proliferation and migration induced by VEGF. Observation of an immunohistochemical analysis of various human tumor tissue arrays revealed that Nrp2 is highly expressed in the tumor vessel lining and to a lesser extent in normal tissue microvessels. From these results, we suggest that Nrp2 may function to increase the response to VEGF, which is more significant in TEC than in NEC given the differential expression, leading to gastric TEC with aggressive angiogenesis phenotypes.

  14. Ectopic expression of single transcription factors directs differentiation of a medaka spermatogonial cell line.

    PubMed

    Thoma, Eva C; Wagner, Toni U; Weber, Isabell P; Herpin, Amaury; Fischer, Andreas; Schartl, Manfred

    2011-08-01

    The capability to form all cell types of the body is a unique feature of stem cells. However, many questions remain concerning the mechanisms regulating differentiation potential. The derivation of spermatogonial cell lines (SGs) from mouse and human, which can differentiate across germ-layer borders, suggested male germ cells as a potential stem cell source in addition to embryonic stem cells. Here, we present a differentiation system using an SG of the vertebrate model organism Oryzias latipes (medaka). We report differentiation of this cell line into 4 different ectodermal and mesodermal somatic cell types. In addition to differentiation into adipocytes by retinoic acid treatment, we demonstrate for the first time that directed differentiation of an SG can be induced by ectopic expression of single transcription factors, completely independent of culture conditions. Transient transfection with mitf-m, a transcription factor that has been shown to induce differentiation into melanocytes in medaka embryonic stem cells, resulted in the formation of the same cell type in spermatogonia. Similarly, the formation of neuron-like cells and matrix-depositing osteoblasts was induced by ectopic expression of mash1 and cbfa1, respectively. Interestingly, we found that the expression of all mentioned fate-inducing transcription factors leads to recapitulation of the temporal pattern of marker gene expression known from in vivo studies. PMID:21090990

  15. Changes in the phenotype of human small cell lung cancer cell lines after transfection and expression of the c-myc proto-oncogene.

    PubMed Central

    Johnson, B E; Battey, J; Linnoila, I; Becker, K L; Makuch, R W; Snider, R H; Carney, D N; Minna, J D

    1986-01-01

    Small cell lung cancer growing in cell culture possesses biologic properties that allow classification into two categories: classic and variant. Compared with classic small cell lung cancer cell lines, variant lines have altered large cell morphology, shorter doubling times, higher cloning efficiencies in soft agarose, and very low levels of L dopa decarboxylase production and bombesin-like immunoreactivity. C-myc is amplified and expressed in some small cell lung cancer cell lines and all c-myc amplified lines studied to date display the variant phenotype. To investigate if c-myc amplification and expression is responsible for the variant phenotype, a normal human c-myc gene was transfected into a cloned classic small cell lung cancer cell line not amplified for or expressing detectable c-myc messenger RNA (mRNA). Clones were isolated with one to six copies of c-myc stably integrated into DNA that expressed c-myc mRNA. In addition, one clone with an integrated neo gene but a deleted c-myc gene was isolated and in this case c-myc was not expressed. C-myc expression in transfected clones was associated with altered large cell morphology, a shorter doubling time, and increased cloning efficiency, but no difference in L dopa decarboxylase levels and bombesin-like immunoreactivity. We conclude increased c-myc expression observed here in transfected clones correlates with some of the phenotypic properties distinguishing c-myc amplified variants from unamplified classic small cell lung cancer lines. Images PMID:3016030

  16. Characterization of a new human melanoma cell line with CD133 expression.

    PubMed

    Gil-Benso, Rosario; Monteagudo, Carlos; Cerdá-Nicolás, Miguel; Callaghan, Robert C; Pinto, Sandra; Martínez-Romero, Alicia; Pellín-Carcelén, Ana; San-Miguel, Teresa; Cigudosa, Juan C; López-Ginés, Concha

    2012-06-01

    A novel human malignant melanoma cell line, designated MEL-RC08, was established from a pericranial metastasis of a malignant melanoma of the skin. The cell line has been subcultured for more than 150 passages and is tumorigenic in nude mice. Growth kinetics, cytogenetics, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control; mutations in BRAF, NRAS, C-KiT, RB, and TP53 genes; and amplification of MDM2, CDK4, and cyclin D1 have been studied. Cytogenetically, the tumor and the cell line showed a hypertriploid karyotype with many clonal numeric and structural abnormalities. DNA flow cytometry showed an aneuploid peak with a DNA index value of 1.5. Mutations in TP53 and BRAF genes were demonstrated in both tumor and cell line. Furthermore, stem cell marker CD133 expression was detected in most cells, together with other stem cell markers, suggesting the presence of cells with tumor-initiating potential in this cell line. PMID:22529031

  17. Functional expression of the serotonin 5-HT7 receptor in human glioblastoma cell lines

    PubMed Central

    Mahé, Cécile; Bernhard, Michel; Bobirnac, Ionel; Keser, Corinna; Loetscher, Erika; Feuerbach, Dominik; Dev, Kumlesh K; Schoeffter, Philippe

    2004-01-01

    Serotonin 5-HT7 receptors are present in astrocytes. Understanding their role in this type of cell would greatly benefit from the identification of astroglial cell lines expressing this receptor type. The aim of the present study was to assess the expression of native 5-HT7 receptors and 5-HT7 receptor mRNA in a number of human glioblastoma cell lines, by means of cAMP measurements, Western blot analysis and reverse transcriptase–polymerase chain reaction (RT–PCR) analysis. 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced concentration-dependent stimulations of cAMP accumulation in the human glioblastoma cell lines, U-373 MG, U-138 MG, U-87 MG, DBTRG-05MG, T98G, H4, CCF-STTG1 and Hs 683. The rank order of potency was 5-CT>5-HT=5-MeOT≫8-OH-DPAT. The effect of 5-CT was inhibited in a concentration-dependent manner by the selective 5-HT7 receptor antagonist SB-269970 in all human glioblastoma cells. Schild analyses yielded slope factors close to unity (0.89–1.13) and pA2 values of 8.69–9.05. Western blot analysis revealed the presence of immunoreactive bands corresponding to the human 5-HT7 receptor in extracts of all human glioblastoma cell lines. The presence of the three splice variants of the 5-HT7 receptor (5-HT7(a/b/d)) was visualized by RT–PCR analysis with specific primers in all human glioblastoma cell lines. In conclusion, human glioblastoma cell lines express functional 5-HT7 receptors and the three splice variants of the corresponding mRNA. These cell lines could serve as model systems of native 5-HT7 receptors in glial cells to investigate their putative role in processes like release of neurotrophic factors or inflammatory cytokines. PMID:15339860

  18. Functional expression of the serotonin 5-HT7 receptor in human glioblastoma cell lines.

    PubMed

    Mahé, Cécile; Bernhard, Michel; Bobirnac, Ionel; Keser, Corinna; Loetscher, Erika; Feuerbach, Dominik; Dev, Kumlesh K; Schoeffter, Philippe

    2004-10-01

    Serotonin 5-HT(7) receptors are present in astrocytes. Understanding their role in this type of cell would greatly benefit from the identification of astroglial cell lines expressing this receptor type. The aim of the present study was to assess the expression of native 5-HT(7) receptors and 5-HT(7) receptor mRNA in a number of human glioblastoma cell lines, by means of cAMP measurements, Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced concentration-dependent stimulations of cAMP accumulation in the human glioblastoma cell lines, U-373 MG, U-138 MG, U-87 MG, DBTRG-05MG, T98G, H4, CCF-STTG1 and Hs 683. The rank order of potency was 5-CT>5-HT=5-MeOT>8-OH-DPAT. The effect of 5-CT was inhibited in a concentration-dependent manner by the selective 5-HT(7) receptor antagonist SB-269970 in all human glioblastoma cells. Schild analyses yielded slope factors close to unity (0.89-1.13) and pA(2) values of 8.69-9.05. Western blot analysis revealed the presence of immunoreactive bands corresponding to the human 5-HT(7) receptor in extracts of all human glioblastoma cell lines. The presence of the three splice variants of the 5-HT(7) receptor (5-HT(7(a/b/d))) was visualized by RT-PCR analysis with specific primers in all human glioblastoma cell lines. In conclusion, human glioblastoma cell lines express functional 5-HT(7) receptors and the three splice variants of the corresponding mRNA. These cell lines could serve as model systems of native 5-HT(7) receptors in glial cells to investigate their putative role in processes like release of neurotrophic factors or inflammatory cytokines. PMID:15339860

  19. Gene expression in cell lines from propionic acidemia patients, carrier parents, and controls.

    PubMed

    Chapman, Kimberly A; Bush, William S; Zhang, Zhe

    2015-08-01

    Propionic acidemia (PA) is an inborn of metabolism which usually presents with metabolic acidosis and accumulation of 3-hydroxypropionate among other toxins. Examining the gene expression in lymphoblastoid cell lines (LCLs) from PA patients, their carrier parents and age/sex-matched controls at normal glucose and low glucose growth conditions demonstrated differences among and between these groups. Using three-way ANOVA analysis, four DAVID clusters of response were identified of which three of the four clusters showed that LCLs from carrier parents had an intermediate response between healthy controls and PA patients. These differences included changes in expression of cell cycle regulatory genes, mitochondrial related genes, and transcriptional regulation. In addition, differences also were observed in expression of genes involved in transendothelial migration and focal adhesion at normal growth conditions when comparing the LCLs from PA patients and controls. These studies demonstrate transcriptional differences between LCLs from PA patients, their parents and biochemically normal controls.

  20. [Four-week simulated weightlessness increases the expression of atrial natriuretic peptide in the myocardium].

    PubMed

    Zhang, Wen-Cheng; Lu, Yuan-Ming; Yang, Huai-Zhang; Xu, Peng-Tao; Chang, Hui; Yu, Zhi-Bin

    2013-04-25

    One of the major circulatory changes that occur in human during space flight and simulated weightlessness is a cerebral redistribution of body fluids, which is accompanied by an increase of blood volume in the upper body. Therefore, atrial myocardium should increase the secretion of atrial natriuretic peptide (ANP), but the researches lack common conclusion until now. The present study was to investigate the expression level of ANP in simulated weightlessness rats, and to confirm the changes of ANP by observing the associated proteins of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The tail-suspended rat model was used to simulate weightlessness. Western blots were carried out to examine the expression levels of ANP and SNARE proteins in atrial and left ventricular myocardium. The results showed that ANP expression in atrial myocardium showed an increase in 4-week tail-suspended rats (SUS) compared with that in the synchronous control rats (CON). We only detected a trace amount of ANP in the left ventricular myocardium of the CON, but found an enhanced expression of ANP in left ventricular myocardium of the SUS. Expression of VAMP-1/2 (vesicle associated SNARE) increased significantly in both atrial and left ventricular myocardium in the SUS compared with that in the CON. There was no difference of the expression of syntaxin-4 (target compartment associated SNARE) between the CON and SUS, but the expression of SNAP-23 showed an increase in atrial myocardium of the SUS compared with that in the CON. Synip and Munc-18c as regulators of SNAREs did not show significant difference between the CON and SUS. These results suggest that the expression of ANP shows an increase in atrial and left ventricular myocardium of 4-week tail-suspended rats. Enhanced expression of VAMP-1/2 associated with ANP vesicles confirms the increased expression of ANP in atrial and left ventricular myocardium.

  1. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines.

    PubMed

    Große-Kreul, Jan; Busch, Maike; Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma. PMID:27626280

  2. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines.

    PubMed

    Große-Kreul, Jan; Busch, Maike; Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma.

  3. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines

    PubMed Central

    Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma. PMID:27626280

  4. Human immunodeficiency virus type 1 Tat increases the expression of cleavage and polyadenylation specificity factor 73-kilodalton subunit modulating cellular and viral expression.

    PubMed

    Calzado, Marco A; Sancho, Rocío; Muñoz, Eduardo

    2004-07-01

    The human immunodeficiency virus type 1 (HIV-1) Tat protein, which is essential for HIV gene expression and viral replication, is known to mediate pleiotropic effects on various cell functions. For instance, Tat protein is able to regulate the rate of transcription of host cellular genes and to interact with the signaling machinery, leading to cellular dysfunction. To study the effect that HIV-1 Tat exerts on the host cell, we identified several genes that were up- or down-regulated in tat-expressing cell lines by using the differential display method. HIV-1 Tat specifically increases the expression of the cleavage and polyadenylation specificity factor (CPSF) 73-kDa subunit (CPSF3) without affecting the expression of the 160- and 100-kDa subunits of the CPSF complex. This complex comprises four subunits and has a key function in the 3'-end processing of pre-mRNAs by a coordinated interaction with other factors. CPSF3 overexpression experiments and knockdown of the endogenous CPSF3 by mRNA interference have shown that this subunit of the complex is an important regulatory protein for both viral and cellular gene expression. In addition to the known CPSF3 function in RNA polyadenylation, we also present evidence that this protein exerts transcriptional activities by repressing the mdm2 gene promoter. Thus, HIV-1-Tat up-regulation of CPSF3 could represent a novel mechanism by which this virus increases mRNA processing, causing an increase in both cell and viral gene expression.

  5. Human Immunodeficiency Virus Type 1 Tat Increases the Expression of Cleavage and Polyadenylation Specificity Factor 73-Kilodalton Subunit Modulating Cellular and Viral Expression

    PubMed Central

    Calzado, Marco A.; Sancho, Rocío; Muñoz, Eduardo

    2004-01-01

    The human immunodeficiency virus type 1 (HIV-1) Tat protein, which is essential for HIV gene expression and viral replication, is known to mediate pleiotropic effects on various cell functions. For instance, Tat protein is able to regulate the rate of transcription of host cellular genes and to interact with the signaling machinery, leading to cellular dysfunction. To study the effect that HIV-1 Tat exerts on the host cell, we identified several genes that were up- or down-regulated in tat-expressing cell lines by using the differential display method. HIV-1 Tat specifically increases the expression of the cleavage and polyadenylation specificity factor (CPSF) 73-kDa subunit (CPSF3) without affecting the expression of the 160- and 100-kDa subunits of the CPSF complex. This complex comprises four subunits and has a key function in the 3′-end processing of pre-mRNAs by a coordinated interaction with other factors. CPSF3 overexpression experiments and knockdown of the endogenous CPSF3 by mRNA interference have shown that this subunit of the complex is an important regulatory protein for both viral and cellular gene expression. In addition to the known CPSF3 function in RNA polyadenylation, we also present evidence that this protein exerts transcriptional activities by repressing the mdm2 gene promoter. Thus, HIV-1-Tat up-regulation of CPSF3 could represent a novel mechanism by which this virus increases mRNA processing, causing an increase in both cell and viral gene expression. PMID:15194760

  6. Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli.

    PubMed

    Kim, Jinyeong; Kim, Seul I; Hong, Eunsoo; Ryu, Yeonwoo

    2016-11-01

    Heterologous proteins expressed in bacteria are used for numerous biotechnological applications. Escherichia coli is the most commonly used host for heterologous protein expression because of its many advantages. Researchers have been studying proteins from extremophiles heterologously expressed in E. coli because the proteins of extremophiles are strongly resistant to extreme conditions. In a previous study, a thermostable esterase Est-AF was isolated from Archaeoglobus fulgidus and expressed in E. coli. However, further studies of Est-AF were difficult owing to its low expression levels in E. coli. In this study, we used various strategies, such as changing the expression vector and host strain, codon optimization, and optimization of induction conditions, to increase the expression of Est-AF. Through codon optimization and by changing the vector and host strain, Est-AF expression was increased from 31.50 ± 0.35 mg/L to 61.75 ± 0.28 mg/L. The optimized expression system consisted of a codon-optimized Est-AF gene in a pET28a(+)-based expression plasmid in E. coli Rosetta cells. The expression level was further increased by optimizing the induction conditions. The optimized conditions were induction with 0.4 mM isopropyl-b-d-1-thiogalactoside (IPTG) at 37 °C for 5 h. Under these conditions, the expression level of Est-AF was increased from 31.5 ± 0.35 mg/L to 119.52 ± 0.34 mg/L. PMID:27449918

  7. Expression and cellular localization of human hyaluronidase-2 in articular chondrocytes and cultured cell lines

    PubMed Central

    Chow, G.; Knudson, C. B.; Knudson, W.

    2011-01-01

    Summary Objective There is debate whether hyaluronan (HA) can be enzymatically degraded within the extracellular matrix of cartilage and other tissues or whether its catabolism occurs strictly within the lysosomal compartment of chondrocytes and other cell types. Previous studies have suggested that one of the lysosomal hyaluronidases (hyaluronidase-2) can be expressed as a functionally-active glycosyl phosphatidylinositol-linked protein at the surface of mammalian cells. If this form of hyaluronidase expression occurs in chondrocytes, this could represent a possible mechanism for extracellular HA cleavage. Thus, which hyaluronidases are expressed and where was the objective of this study. Methods mRNA for hyaluronidases was quantified by reverse transcription-polymerase chain reaction (RT-PCR) and enzymatic activity by HA zymograms. Recombinant forms of hyaluronidase-2 were generated and expressed in model cell lines. A peptide-specific polyclonal antiserum was prepared to localize endogenous human hyaluronidase-2 in human articular chondrocytes. Results Hyaluronidase-2 is the principal mRNA transcript expressed by primary human articular chondrocytes as well as various model cell lines. Recombinant hyaluronidase-2, containing N-terminal or C-terminal epitope tags, was strictly localized intracellularly and not released by treatment with a phosphatidylinositol-specific phospholipase. Endogenous hyaluronidase-2 expressed by human chondrocytes as well as HeLa cells could only be detected following detergent permeabilization of the plasma membranes. Conclusions These data suggest that on chondrocytes and other cell types examined, hyaluronidase-2 is not present or functional at the external plasma membrane. Thus, local turnover of HA is dependent on receptor-mediated endocytosis and delivery to low pH intracellular organelles for its complete degradation. PMID:16600643

  8. Genetic and epigenetic factors affect RET gene expression in breast cancer cell lines and influence survival in patients

    PubMed Central

    Griseri, Paola; Garrone, Ornella; Lo Sardo, Alessandra; Monteverde, Martino; Rusmini, Marta; Tonissi, Federica; Merlano, Marco; Bruzzi, Paolo

    2016-01-01

    Germline and somatic mutations play a crucial role in breast cancer (BC), driving the initiation, progression, response to therapy and outcome of the disease. Hormonal therapy is limited to patients with tumors expressing steroid hormone receptors, such as estrogen receptor (ER), nevertheless resistance often limits its success. The RET gene is known to be involved in neurocristopathies such as Hirschsprung disease and Multiple Endocrine Neoplasia type 2, in the presence of loss-of-function and gain-of-function mutations, respectively. More recently, RET over-expression has emerged as a new player in ER-positive (ER+) BC, and as a potential target to enhance sensitivity and avoid resistance to tamoxifen therapy. Therefore, targeting the RET pathway may lead to new therapies in ER+ BC. To this end, we have investigated the molecular mechanisms which underlie RET overexpression and its possible modulation in two BC cell lines, MCF7 and T47D, showing different RET expression levels. Moreover, we have carried out a pilot association study in 93 ER+ BC patients. Consistent with the adverse role of RET over-expression in BC, increased overall survival was observed in carriers of the variant allele of SNP rs2435357, a RET polymorphism already known to be associated with reduced RET expression. PMID:27034161

  9. NanoCAGE analysis of the mouse olfactory epithelium identifies the expression of vomeronasal receptors and of proximal LINE elements

    PubMed Central

    Pascarella, Giovanni; Lazarevic, Dejan; Plessy, Charles; Bertin, Nicolas; Akalin, Altuna; Vlachouli, Christina; Simone, Roberto; Faulkner, Geoffrey J.; Zucchelli, Silvia; Kawai, Jun; Daub, Carsten O.; Hayashizaki, Yoshihide; Lenhard, Boris; Carninci, Piero; Gustincich, Stefano

    2013-01-01

    By coupling laser capture microdissection to nanoCAGE technology and next-generation sequencing we have identified the genome-wide collection of active promoters in the mouse Main Olfactory Epithelium (MOE). Transcription start sites (TSSs) for the large majority of Olfactory Receptors (ORs) have been previously mapped increasing our understanding of their promoter architecture. Here we show that in our nanoCAGE libraries of the mouse MOE we detect a large number of tags mapped in loci hosting Type-1 and Type-2 Vomeronasal Receptors genes (V1Rs and V2Rs). These loci also show a massive expression of Long Interspersed Nuclear Elements (LINEs). We have validated the expression of selected receptors detected by nanoCAGE with in situ hybridization, RT-PCR and qRT-PCR. This work extends the repertory of receptors capable of sensing chemical signals in the MOE, suggesting intriguing interplays between MOE and VNO for pheromone processing and positioning transcribed LINEs as candidate regulatory RNAs for VRs expression. PMID:24600346

  10. [Expression of MicroRNAs of An Interneuron Precursor Cell Line GE6 in Various Differentiation Conditions].

    PubMed

    Ge, Xinxu; Liu, Qian; Yin, Shu; Li, Hedong

    2015-12-01

    The purpose of this study was to identify specific microRNAs (miRNAs) during differentiation and maturation of interneurons and to predict their possible functions by analyzing the expression of miRNAs during in vitro differentiation of the rat interneuron precursor cell line GE6. In the experiment, the interneuron precursor cell line GE6 was cultured under three different conditions, i. e. the first was that had not added growth factors and the normal differentiation cultured for 4 days (Ge6_4d); the second was that cultured with bone morphogenetic protein-2 (BMP2) for 4 days (Ge6_bmp2); and the third was that cultured with sonic hedgehog (SHH) for 4 days (Ge6_ shh). In addition, another group of undifferentiated GE6 (Ge6_u) was applied as a control. We found in this study that the expression levels of a large number of miRNAs changed significantly during GE6 differentiation. The expression levels of miR-710, miR-290-5p and miR-3473 increased in the GE6 cells with secreted factor BMP2. These miRNAs may play important regulatory roles during interneuron differentiation. PMID:27079100

  11. Increased expression of the Ras suppressor Rsu-1 enhances Erk-2 activation and inhibits Jun kinase activation.

    PubMed

    Masuelli, L; Cutler, M L

    1996-10-01

    Studies were undertaken to determine the effect of the Ras suppressor Rsu-1 on Ras signal transduction pathways in two different cell backgrounds. An expression vector containing the mouse rsu-1 cDNA under the control of a mouse mammary tumor virus promoter was introduced into NIH 3T3 cells and the pheochromocytoma cell line PC12. Cell lines developed in the NIH 3T3 background expressed p33rsu-1 at approximately twice the normal endogenous level. However, PC12 cell clones which expressed p33rsu-1 at an increased level in a regulatable fashion in response to dexamethasone were isolated. Analysis of proteins involved in regulation of Ras and responsive to Ras signal transduction revealed similar changes in the two cell backgrounds in the presence of elevated p33rsu-1. There was an increase in the level of SOS, the guanine nucleotide exchange factor, and an increase in the percentage of GTP-bound Ras. In addition, there was an increase in the amount of p120 Ras-specific GTPase-activating protein (GAP) and GAP-associated p190. However, a decrease in Ras GTPase-activating activity was detected in lysates of the Rsu-1 transfectants, and immunoprecipitated p120 GAP from the Rsu-1 transfectants showed less Ras GTPase-activating activity than GAP from control cells. Activation of Erk-2 kinase by growth factor and tetradecanyol phorbol acetate was greater in the Rsu-1 transfectants than in control cells. However, c-Jun amino-terminal kinase activity (Jun kinase) was not activatable by epidermal growth factor in Rsu-1 PC12 cell transfectants, in contrast to the PC12 vector control cell line. Transient expression of p33rsu-1 in Cos1 cells following cotransfection with either hemagglutinin-tagged Jun kinase or hemagglutinin-tagged Erk-2 revealed that Rsu-1 expression inhibited constitutive Jun kinase activity while enhancing Erk-2 activity. Detection of in vitro binding of Rsu-1 to Raf-1 suggested that in Rsu-1 transfectants, increased activation of the Raf-1 pathway occurred

  12. Calpain activity and expression are increased in splenic inflammatory cells associated with experimental allergic encephalomyelitis.

    PubMed

    Shields, D C; Schaecher, K E; Goust, J M; Banik, N L

    1999-09-01

    Since calcium-activated neutral proteinase (calpain) activity and expression are significantly increased in activated glial/inflammatory cells in the central nervous system of animals with autoimmune demyelinating diseases, this enzyme may also play a role in peripheral organ systems in these diseases. In this study, the activity and expression of calpain and the endogenous inhibitor, calpastatin, were evaluated at transcriptional and translational levels in spleens of Lewis rats with acute experimental allergic encephalomyelitis (EAE) prior to the onset of clinical symptoms. Calpain activity and translational expression were increased by 475.5% and 44.3% respectively, on day 4 post-induction in adjuvant controls and animals with EAE. These levels remained elevated compared to normal controls on days 8 and 12. Calpastatin translational expression was similarly increased at these time points although transcriptional expression was not significantly altered at any time following induction of EAE. Likewise, transcriptional expression of mu-calpain was unchanged following induction, while small increases in m-calpain transcriptional expression were observed on days 2 and 8. Most calpain expression was observed in activated splenic macrophages at day 8 post-induction even though activated T cells were also calpain positive. In spinal cords of animals with EAE, calpain expression was significantly increased in rats with severe disease compared to those exhibiting only mild symptoms at day 12 post-induction. Thus, prior to symptomatic EAE, increased calpain activity and expression in peripheral lymphoid organs may play an important role in T cell migration and subsequent disease progression. PMID:10496171

  13. Botulinum neurotoxin type A inhibits synaptic vesicle 2 expression in breast cancer cell lines

    PubMed Central

    Bandala, C; Cortés-Algara, AL; Mejía-Barradas, CM; Ilizaliturri-Flores, I; Dominguez-Rubio, R; Bazán-Méndez, CI; Floriano-Sánchez, E; Luna-Arias, JP; Anaya-Ruiz, M; Lara-Padilla, E

    2015-01-01

    Aim: It is known that botulinum neurotoxin type A (BoNTA) improves some kinds of cancer (e.g. prostate) and that synaptic vesicle glycoprotein 2 (SV2) is the molecular target of this neurotoxin. Besides having potential therapeutic value, this glycoprotein has recently been proposed as a molecular marker for several types of cancer. Although the mechanisms of cancer development and the improvement found with botulinum treatment are not well understood, the formation of the botulinum-SV2 complex may influence the presence and distribution of SV2 and the function of vesicles. To date, there are no reports on the possible effect of botulinum on breast cancer of unknown causes, which have a great impact on women’s health. Thus we determined the presence of SV2 in three breast cancer cell lines and the alterations found with botulinum application. Materials and methods: With and without adding 10 units of botulinum, SV2 protein expression was determined by optical densitometry in T47D, MDA-MB-231 and MDA-MB-453 cell lines and the distribution of SV2 was observed with immunochemistry (hematoxylin staining). Results: The SV2 protein was abundant in the cancer cells herein tested, and maximally so in T47D. In all three cancer cell lines botulinum diminished SV2 expression, which was found mostly in the cell periphery. Conclusion: SV2 could be a molecular marker in breast cancer. Its expression and distribution is regulated by botulinum, suggesting an interesting control mechanism for SV2 expression and a possible alternative therapy. Further studies are needed in this sense. PMID:26339411

  14. Aerobic conditions increase isoprenoid biosynthesis pathway gene expression levels for carotenoid production in Enterococcus gilvus.

    PubMed

    Hagi, Tatsuro; Kobayashi, Miho; Nomura, Masaru

    2015-06-01

    Some lactic acid bacteria that harbour carotenoid biosynthesis genes (crtNM) can produce carotenoids. Although aerobic conditions can increase carotenoid production and crtNM expression levels, their effects on the pathways that synthesize carotenoid precursors such as mevalonate and isoprene are not completely understood. In this study, we investigated whether aerobic conditions affected gene expression levels involved in the isoprenoid biosynthesis pathway that includes the mevalonate and isoprene biosynthesis pathways in Enterococcus gilvus using real-time quantitative reverse transcription PCR. NADH oxidase (nox) and superoxide dismutase (sod) gene expression levels were investigated as controls for aerobic conditions. The expression levels of nox and sod under aerobic conditions were 7.2- and 8.0-fold higher, respectively, than those under anaerobic conditions. Aerobic conditions concomitantly increased the expression levels of crtNM carotenoid biosynthesis genes. HMG-CoA synthase gene expression levels in the mevalonate pathway were only slightly increased under aerobic conditions, whereas the expression levels of HMG-CoA reductase and five other genes in the isoprene biosynthesis pathways were 1.2-2.3-fold higher than those under anaerobic conditions. These results demonstrated that aerobic conditions could increase the expression levels of genes involved in the isoprenoid biosynthesis pathway via mevalonate in E. gilvus.

  15. Increased gastrin gene expression provides a physiological advantage to mice under hypoxic conditions.

    PubMed

    Laval, Marie; Baldwin, Graham S; Shulkes, Arthur; Marshall, Kathryn M

    2015-01-15

    Hypoxia, or a low concentration of O2, is encountered in humans undertaking activities such as mountain climbing and scuba diving and is important pathophysiologically as a limiting factor in tumor growth. Although data on the interplay between hypoxia and gastrins are limited, gastrin expression is upregulated by hypoxia in gastrointestinal cancer cell lines, and gastrins counterbalance hypoxia by stimulating angiogenesis in vitro and in vivo. The aim of this study was to determine if higher concentrations of the gastrin precursor progastrin are protective against hypoxia in vivo. hGAS mice, which overexpress progastrin in the liver, and mice of the corresponding wild-type FVB/N strain were exposed to normoxia or hypoxia. Iron status was assessed by measurement of serum iron parameters, real-time PCR for mRNAs encoding critical iron regulatory proteins, and Perls' stain and atomic absorption spectrometry for tissue iron concentrations. FVB/N mice lost weight at a faster rate and had higher sickness scores than hGAS mice exposed to hypoxia. Serum iron levels were lower in hGAS than FVB/N mice and decreased further when the animals were exposed to hypoxia. The concentration of iron in the liver was strikingly lower in hGAS than FVB/N mice. We conclude that increased circulating concentrations of progastrin provide a physiological advantage against systemic hypoxia in mice, possibly by increasing the availability of iron stores. This is the first report of an association between progastrin overexpression, hypoxia, and iron homeostasis. PMID:25394662

  16. ErbB2 Signaling Increases Androgen Receptor Expression in Abiraterone-Resistant Prostate Cancer

    PubMed Central

    Gao, Shuai; Ye, Huihui; Gerrin, Sean; Wang, Hongyun; Sharma, Ankur; Chen, Sen; Patnaik, Akash; Sowalsky, Adam G.; Voznesensky, Olga; Han, Wanting; Yu, Ziyang; Mostaghel, Elahe A.; Nelson, Peter S.; Taplin, Mary-Ellen; Balk, Steven P.; Cai, Changmeng

    2016-01-01

    Purpose ErbB2 signaling appears to be increased and may enhance AR activity in a subset of CRPC, but agents targeting ErbB2 have not been effective. This study was undertaken to assess ErbB2 activity in abiraterone-resistant prostate cancer (PCa), and determine whether it may contribute to androgen receptor (AR) signaling in these tumors. Experimental Design AR activity and ErbB2 signaling were examined in the radical prostatectomy specimens from a neoadjuvant clinical trial of leuprolide plus abiraterone, and in the specimens from abiraterone-resistant CRPC xenograft models. The effect of ErbB2 signaling on AR activity was determined in two CRPC cell lines. Moreover, the effect of combination treatment with abiraterone and an ErbB2 inhibitor was assessed in a CRPC xenograft model. Results We found that ErbB2 signaling was elevated in residual tumor following abiraterone treatment in a subset of patients, and was associated with higher nuclear AR expression. In xenograft models, we similarly demonstrated that ErbB2 signaling was increased and associated with AR reactivation in abiraterone-resistant tumors. Mechanistically, we show that ErbB2 signaling and subsequent activation of the PI3K/AKT signaling stabilizes AR protein. Furthermore, concomitantly treating CRPC cells with abiraterone and an ErbB2 inhibitor, lapatinib, blocked AR reactivation and suppressed tumor progression. Conclusions ErbB2 signaling is elevated in a subset of abiraterone-resistant prostate cancer patients and stabilizes AR protein. Combination therapy with abiraterone and ErbB2 antagonists may be effective for treating the subset of CRPC with elevated ErbB2 activity. PMID:26936914

  17. Lines

    ERIC Educational Resources Information Center

    Mires, Peter B.

    2006-01-01

    National Geography Standards for the middle school years generally stress the teaching of latitude and longitude. There are many creative ways to explain the great grid that encircles our planet, but the author has found that students in his college-level geography courses especially enjoy human-interest stories associated with lines of latitude…

  18. HO-1 expression increases mesenchymal stem cell-derived osteoblast but decreases adipocyte lineage

    PubMed Central

    Vanella, Luca; Kim, Dong Hyun; Asprinio, David; Peterson, Stephen J.; Barbagallo, Ignazio; Vanella, Angelo; Goldstein, Dove; Ikehara, Susumu; Abraham, Nader G.

    2009-01-01

    Human bone marrow mesenchymal stem cells (MSC) are pleitrophic cells that differentiate to either adipocytes or osteoblasts as a result of cross-talk by specific signaling pathways including heme oxygenase (HO)-1/-2 expression. We examined the effect of inducers of HO-1 expression and inhibitors of HO activity on MSC differentiation to the osteoblast and adipogenesis lineage. HO-1 expression is increased during osteoblast stem cell development, but remains elevated, at 25 days. The increase in HO-1 levels proceed an increase in alkaline phosphatase (AP) activity and an increase in BMP, osteonectin and RUNX-2 mRNA. Induction of HO-1 by osteogenic growth peptide (OGP) was associated with an increase in BMP-2 and osteonectin. Exposure of MSC to high glucose levels decreased osteocalcin and osteogenic protein expression, which was reversed by upregulation of the OGP-mediated increase in HO-1 expression. The glucose mediated decrease in HO-1 resulted in decreased levels of pAMPK, pAKT and the eNOS signaling pathway and was reversed by OGP. In contrast, MSC-derived adipocytes were increased by glucose. HO-1 siRNA decreased HO-1 expression but increased adipocyte stem cell differentiation and the adipogenesis marker, PPARγ. Thus, upregulation of HO-1 expression shifts the balance of MSC differentiation in favor of the osteoblast lineage. In contrast, a decrease in HO-1 or exposure to glucose drives the MSC towards adipogenesis. Thus targeting HO-1 expression is a portal to increased osteoblast stem cell differentiation and to the attenuation of osteoporosis by the promotion of bone formation. PMID:19853072

  19. TNF-α increases endothelial progenitor cell adhesion to the endothelium by increasing bond expression and affinity

    PubMed Central

    Prisco, Anthony R.; Prisco, Michael R.; Carlson, Brian E.

    2014-01-01

    Endothelial progenitor cells (EPCs) are a rare population of cells that participate in angiogenesis. To effectively use EPCs for regenerative therapy, the mechanisms by which they participate in tissue repair must be elucidated. This study focused on the process by which activated EPCs bind to a target tissue. It has been demonstrated that EPCs can bind to endothelial cells (ECs) through the tumore necrosis factor-α (TNF-α)-regulated vascular cell adhesion molecule 1/very-late antigen 4 (VLA4) interaction. VLA4 can bind in a high or low affinity state, a process that is difficult to experimentally isolate from bond expression upregulation. To separate these processes, a new parallel plate flow chamber was built, a detachment assay was developed, and a mathematical model was created that was designed to analyze the detachment assay results. The mathematical model was developed to predict the relative expression of EPC/EC bonds made for a given bond affinity distribution. EPCs treated with TNF-α/vehicle were allowed to bind to TNF-α/vehicle-treated ECs in vitro. Bound cells were subjected to laminar flow, and the cellular adherence was quantified as a function of shear stress. Experimental data were fit to the mathematical model using changes in bond expression or affinity as the only free parameter. It was found that TNF-α treatment of ECs increased adhesion through bond upregulation, whereas TNF-α treatment of EPCs increased adhesion by increasing bond affinity. These data suggest that injured tissue could potentially increase recruitment of EPCs for tissue regeneration via the secretion of TNF-α. PMID:25539711

  20. Transepithelial resistance and claudin expression in trout RTgill-W1 cell line: effects of osmoregulatory hormones.

    PubMed

    Trubitt, Rebecca T; Rabeneck, D Brett; Bujak, Joanna K; Bossus, Maryline C; Madsen, Steffen S; Tipsmark, Christian K

    2015-04-01

    In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1-2 days (20-80Ω⋅cm(2)), which was then maintained for more than 6 days. Tetrabromocinnamic acid, a known stimulator of TER via casein kinase II inhibition, elevated TER in the cell line to 125% of control values after 2 and 6h. Treatment with ethylenediaminetetraacetic acid induced a decrease in TER to <15% of pre-treatment level. Cortisol elevated TER after 12-72 h in a concentration-dependent manner, and this increase was antagonized by growth hormone (Gh). The effects of three osmoregulatory hormones, Gh, prolactin, and cortisol, on the mRNA expression of three tight junction proteins were examined: claudin-10e (Cldn-10e), Cldn-30, and zonula occludens-1 (Zo-1). The expression of cldn-10e was stimulated by all three hormones but with the strongest effect of Gh (50-fold). cldn-30 expression was stimulated especially by cortisol (20-fold) and also by Gh (4-fold). Finally, zo-1 was unresponsive to hormone treatment. Western blot analysis detected Cldn-10e and Cldn-30 immunoreactive proteins of expected molecular weight in samples from rainbow trout gills but not from RTgill-W1 cultures, possibly due to low expression levels. Collectively, these results show that the RTgill-W1 cell layers have tight junctions between cells, are sensitive to hormone treatments, and may provide a useful model for in vitro study of some in vivo gill phenomena.

  1. Neurotensin Decreases the Proinflammatory Status of Human Skin Fibroblasts and Increases Epidermal Growth Factor Expression

    PubMed Central

    Miguel Neves, Bruno; Cruz, Maria Teresa; Carvalho, Eugénia

    2014-01-01

    Fibroblasts colonization into injured areas during wound healing (WH) is responsible for skin remodelling and is also involved in the modulation of inflammation, as fibroblasts are immunologically active. Herein, we aimed to determine neurotensin effect on the immunomodulatory profile of fibroblasts, both in homeostatic and inflammatory conditions. Neurotensin mediated responses occurred through NTR1 or NTR3 receptors, while under inflammatory conditions NTR1 expression increase seemed to modulate neurotensin responses. Among different immunomodulatory genes, CCL11, IL-8, and IL-6 were the most expressed genes, while CCL4 and EGF were the less expressed genes. After neurotensin exposure, IL-8 mRNA expression was increased while CCL11 was decreased, suggesting a proinflammatory upregulation and chemoattractant ability downregulation of fibroblasts. Under inflammatory conditions, gene expression was significantly increased. After neurotensin exposure, CCL4 and IL-6 mRNA expression were decreased while CCL11 was increased, suggesting again a decrease in the chemoattractant capacity of fibroblasts and in their proinflammatory status. Furthermore, the expression of EGF, a crucial growth factor for skin cells proliferation and WH, was increased in all conditions. Overall, neurotensin, released by nerve fibers or skin cells, may be involved in the decrease of the chemotaxis and the proinflammatory status in the proliferation and remodelling phases of WH. PMID:25180119

  2. Increased Gene Expression by the First Intron of Maize Shrunken-1 Locus in Grass Species 1

    PubMed Central

    Vasil, Vimla; Clancy, Maureen; Ferl, Robert J.; Vasil, Indra K.; Hannah, L. Curtis

    1989-01-01

    The first intron of the shrunken-1 (Sh1) locus of maize was incorporated into constructs containing the chloramphenicol acetyltransferase gene (CAT) coupled with the nopaline synthase 3′ polyadenylation signal. Transcription was driven with the 35S promoter of the cauliflower mosaic virus (CaMV) or the Sh1 promoter of maize. Transient gene expression was monitored following electroporation into protoplasts of Panicum maximum (guineagrass), Pennisetum purpureum (napiergrass), or Zea mays (maize). The 1028 base pair intron increased gene expression in cells of each species when transcription was driven with the 35S promoter. Eleven to 91-fold increases were observed. Expression levels observed in maize were two and eight times those observed in napiergrass and guineagrass, respectively. The 35S promoter gave CAT activity 10 to 100 times that observed with the Sh1 promoter. Whereas expression driven by the 35S promoter was reproducible, that observed with the Sh1 promoter proved quite variable. In similar constructs the first intron of the alcohol dehydrogenase-1 (Adh1) gene of maize led to increased gene expression of only 7 to 10% of that observed with the Sh1 first intron. The increased level of gene expression caused by the Sh1 first intron is approximately 10 times higher than that caused by any other plant introns that have been used. Thus, the Sh1 first intron may prove quite useful in increasing expression of foreign genes in monocots and possibly other plants. Images Figure 2 PMID:16667219

  3. Neurotensin decreases the proinflammatory status of human skin fibroblasts and increases epidermal growth factor expression.

    PubMed

    Pereira da Silva, Lucília; Miguel Neves, Bruno; Moura, Liane; Cruz, Maria Teresa; Carvalho, Eugénia

    2014-01-01

    Fibroblasts colonization into injured areas during wound healing (WH) is responsible for skin remodelling and is also involved in the modulation of inflammation, as fibroblasts are immunologically active. Herein, we aimed to determine neurotensin effect on the immunomodulatory profile of fibroblasts, both in homeostatic and inflammatory conditions. Neurotensin mediated responses occurred through NTR1 or NTR3 receptors, while under inflammatory conditions NTR1 expression increase seemed to modulate neurotensin responses. Among different immunomodulatory genes, CCL11, IL-8, and IL-6 were the most expressed genes, while CCL4 and EGF were the less expressed genes. After neurotensin exposure, IL-8 mRNA expression was increased while CCL11 was decreased, suggesting a proinflammatory upregulation and chemoattractant ability downregulation of fibroblasts. Under inflammatory conditions, gene expression was significantly increased. After neurotensin exposure, CCL4 and IL-6 mRNA expression were decreased while CCL11 was increased, suggesting again a decrease in the chemoattractant capacity of fibroblasts and in their proinflammatory status. Furthermore, the expression of EGF, a crucial growth factor for skin cells proliferation and WH, was increased in all conditions. Overall, neurotensin, released by nerve fibers or skin cells, may be involved in the decrease of the chemotaxis and the proinflammatory status in the proliferation and remodelling phases of WH. PMID:25180119

  4. Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line

    PubMed Central

    Zhuge, Jian; Luo, Ye; Yu, Ying-Nian

    2003-01-01

    AIM: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many small-molecule substrates and volatile anesthetics. CYP2E1 is involved in chemical activation of many carcinogens, procarcinogens, and toxicants. To assess the metabolic and toxicological characteristics of CYP2E1, we cloned CYP2E1 cDNA and established a HepG2 cell line stably expressing recombinant CYP 2E1. METHODS: Human CYP2E1 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2E1 to HepG2 cells. The expression of CYP2E1 mRNA was validated by RT-PCR. The enzyme activity of CYP2E1 catalyzing oxidation of 4-nitrophenol in postmitochondrial supernate (S9) fraction of the cells was determined by spectrophotometry. The metabolic activation of HepG2-CYP2E1 cells was assayed by N-nitrosodiethylamine (NDEA) cytotoxicity and micronucleus test. RESULTS: The cloned CYP2E1 cDNA segment was identical to that reported by Umeno et al (GenBank access No. J02843). HepG2-CYP2E1 cells expressed CYP2E1 mRNA and had 4-nitrophenol hydroxylase activity (0.162 ± 0.025 nmol·min-1·mg-1 S9 protein), which were undetectable in parent HepG2 cells. HepG2-CYP2E1 cells increased the cytotoxicity and micronucleus rate of NDEA in comparison with those of HepG2 cells. CONCLUSION: The cDNA of human CYP2E1 can be successfully cloned, and a cell line, HepG2-CYP2E1, which can efficiently express mRNA and has CYP2E1 activity, is established. The cell line is useful for testing the cytotoxicity, mutagenicity and metabolism of xenobiotics, which may possibly be activated or metabolized by CYP2E1. PMID:14669323

  5. Establishment of radioactive astatine and iodine uptake in cancer cell lines expressing the human sodium/iodide symporter.

    PubMed

    Petrich, T; Helmeke, H-J; Meyer, G J; Knapp, W H; Pötter, E

    2002-07-01

    The sodium/iodide symporter (NIS) has been recognized as an attractive target for radioiodine-mediated cancer gene therapy. In this study we investigated the role of human NIS for cellular uptake of the high LET alpha-emitter astatine-211 ((211)At) in comparison with radioiodine as a potential radionuclide for future applications. A mammalian NIS expression vector was constructed and used to generate six stable NIS-expressing cancer cell lines (three derived from thyroid carcinoma, two from colon carcinoma, one from glioblastoma). Compared with the respective control cell lines, steady state radionuclide uptake of NIS-expressing cell lines increased up to 350-fold for iodine-123 ((123)I), 340-fold for technetium-99m pertechnetate ((99m)TcO(4)(-)) and 60-fold for (211)At. Cellular (211)At accumulation was found to be dependent on extracellular Na(+) ions and displayed a similar sensitivity towards sodium perchlorate inhibition as radioiodide and (99m)TcO(4)(-) uptake. Heterologous competition with unlabelled NaI decreased NIS-mediated (211)At uptake to levels of NIS-negative control cells. Following uptake both radioiodide and (211)At were rapidly (apparent t(1/2) 3-15 min) released by the cells as determined by wash-out experiments. Data of scintigraphic tumour imaging in a xenograft nude mice model of transplanted NIS-modified thyroid cells indicated that radionuclide uptake in NIS-expressing tumours was up to 70 times ((123)I), 25 times ((99m)TcO(4)(-)) and 10 times ((211)At) higher than in control tumours or normal tissues except stomach (3-5 times) and thyroid gland (5-10 times). Thirty-four percent and 14% of the administered activity of (123)I and (211)At, respectively, was found in NIS tumours by region of interest analysis ( n=2). Compared with cell culture experiments, the effective half-life in vivo was greatly prolonged (6.5 h for (123)I, 5.2 h for (211)At) and preliminary dosimetric calculations indicate high tumour absorbed doses (3.5 Gy

  6. Establishment of radioactive astatine and iodine uptake in cancer cell lines expressing the human sodium/iodide symporter.

    PubMed

    Petrich, T; Helmeke, H-J; Meyer, G J; Knapp, W H; Pötter, E

    2002-07-01

    The sodium/iodide symporter (NIS) has been recognized as an attractive target for radioiodine-mediated cancer gene therapy. In this study we investigated the role of human NIS for cellular uptake of the high LET alpha-emitter astatine-211 ((211)At) in comparison with radioiodine as a potential radionuclide for future applications. A mammalian NIS expression vector was constructed and used to generate six stable NIS-expressing cancer cell lines (three derived from thyroid carcinoma, two from colon carcinoma, one from glioblastoma). Compared with the respective control cell lines, steady state radionuclide uptake of NIS-expressing cell lines increased up to 350-fold for iodine-123 ((123)I), 340-fold for technetium-99m pertechnetate ((99m)TcO(4)(-)) and 60-fold for (211)At. Cellular (211)At accumulation was found to be dependent on extracellular Na(+) ions and displayed a similar sensitivity towards sodium perchlorate inhibition as radioiodide and (99m)TcO(4)(-) uptake. Heterologous competition with unlabelled NaI decreased NIS-mediated (211)At uptake to levels of NIS-negative control cells. Following uptake both radioiodide and (211)At were rapidly (apparent t(1/2) 3-15 min) released by the cells as determined by wash-out experiments. Data of scintigraphic tumour imaging in a xenograft nude mice model of transplanted NIS-modified thyroid cells indicated that radionuclide uptake in NIS-expressing tumours was up to 70 times ((123)I), 25 times ((99m)TcO(4)(-)) and 10 times ((211)At) higher than in control tumours or normal tissues except stomach (3-5 times) and thyroid gland (5-10 times). Thirty-four percent and 14% of the administered activity of (123)I and (211)At, respectively, was found in NIS tumours by region of interest analysis ( n=2). Compared with cell culture experiments, the effective half-life in vivo was greatly prolonged (6.5 h for (123)I, 5.2 h for (211)At) and preliminary dosimetric calculations indicate high tumour absorbed doses (3.5 Gy

  7. Curcumin down-regulates AR gene expression and activation in prostate cancer cell lines.

    PubMed

    Nakamura, Keiichiro; Yasunaga, Yutaka; Segawa, Takehiko; Ko, Daejin; Moul, Judd W; Srivastava, Shiv; Rhim, Johng S

    2002-10-01

    Curcumin, traditionally used as a seasoning spice in Indian cuisine, has been reported to decrease the proliferation potential of prostate cancer cells, by a mechanism that is not fully understood. In the current study, we have evaluated the effects of curcumin in cell growth, activation of signal transduction, and transforming activities of both androgen-dependent and independent cell lines. Prostate cancer cell lines, LNCaP and PC-3, were treated with curcumin and its effects were further analyzed on signal transduction and expression of androgen receptor (AR) and AR-related cofactors using transient transfection assay and Western blotting. Our results show that curcumin down-regulates transactivation and expression of AR, activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), and CREB (cAMP response element-binding protein)-binding protein (CBP). Curcumin also inhibited the transforming activities of both cell lines as evidenced by the reduced colony forming ability in soft agar. The results obtained here demonstrate that curcumin has a potential therapeutic effect on prostate cancer cells through down-regulation of AR and AR-related cofactors (AP-1, NF-kappaB and CBP). PMID:12239622

  8. Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells.

    PubMed

    Walter, B A; Purmessur, D; Moon, A; Occhiogrosso, J; Laudier, D M; Hecht, A C; Iatridis, J C

    2016-01-01

    The mechanical behaviour and cellular metabolism of intervertebral discs (IVDs) and articular cartilage are strongly influenced by their proteoglycan content and associated osmotic properties. This osmotic environment is a biophysical signal that changes with disease and may contribute to the elevated matrix breakdown and altered biologic response to loading observed in IVD degeneration and osteoarthritis. This study tested the hypothesis that changes in osmo-sensation by the transient receptor potential vallinoid-4 (TRPV4) ion channel occur with disease and contribute to the inflammatory environment found during degeneration. Immunohistochemistry on bovine IVDs from an inflammatory organ culture model were used to investigate if TRPV4 is expressed in the IVD and how expression changes with degeneration. Western blot, live-cell calcium imaging, and qRT-PCR were used to investigate whether osmolarity changes or tumour necrosis factor α (TNFα) regulate TRPV4 expression, and how altered TRPV4 expression influences calcium signalling and pro-inflammatory cytokine expression. TRPV4 expression correlated with TNFα expression, and was increased when cultured in reduced medium osmolarity and unaltered with TNFα-stimulation. Increased TRPV4 expression increased the calcium flux following TRPV4 activation and increased interleukin-1β (IL-1β) and IL-6 gene expression in IVD cells. TRPV4 expression was qualitatively elevated in regions of aggrecan depletion in degenerated human IVDs. Collectively, results suggest that reduced tissue osmolarity, likely following proteoglycan degradation, can increase TRPV4 signalling and enhance pro-inflammatory cytokine production, suggesting changes in TRPV4 mediated osmo-sensation may contribute to the progressive matrix breakdown in disease. PMID:27434269

  9. REDUCED TISSUE OSMOLARITY INCREASES TRPV4 EXPRESSION AND PRO-INFLAMMATORY CYTOKINES IN INTERVERTEBRAL DISC CELLS

    PubMed Central

    Walter, B.A.; Purmessur, D; Moon, A.; Occhiogrosso, J.; Laudier, D.M.; Hecht, A.C.; Iatridis, J.C.

    2016-01-01

    The mechanical behaviour and cellular metabolism of intervertebral discs (IVDs) and articular cartilage are strongly influenced by their proteoglycan content and associated osmotic properties. This osmotic environment is a biophysical signal that changes with disease and may contribute to the elevated matrix breakdown and altered biologic response to loading observed in IVD degeneration and osteoarthritis. This study tested the hypothesis that changes in osmo-sensation by the transient receptor potential vallinoid-4 (TRPV4) ion channel occur with disease and contribute to the inflammatory environment found during degeneration. Immunohistochemistry on bovine IVDs from an inflammatory organ culture model were used to investigate if TRPV4 is expressed in the IVD and how expression changes with degeneration. Western blot, live-cell calcium imaging, and qRT-PCR were used to investigate whether osmolarity changes or tumour necrosis factor α (TNFα) regulate TRPV4 expression, and how altered TRPV4 expression influences calcium signalling and pro-inflammatory cytokine expression. TRPV4 expression correlated with TNFα expression, and was increased when cultured in reduced medium osmolarity and unaltered with TNFα-stimulation. Increased TRPV4 expression increased the calcium flux following TRPV4 activation and increased interleukin-1β (IL-1β) and IL-6 gene expression in IVD cells. TRPV4 expression was qualitatively elevated in regions of aggrecan depletion in degenerated human IVDs. Collectively, results suggest that reduced tissue osmolarity, likely following proteoglycan degradation, can increase TRPV4 signalling and enhance pro-inflammatory cytokine production, suggesting changes in TRPV4 mediated osmo-sensation may contribute to the progressive matrix breakdown in disease. PMID:27434269

  10. NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression

    PubMed Central

    Spina, Anna; Montella, Roberta; Liccardo, Davide; De Rosa, Alfredo; Laino, Luigi; Mitsiadis, Thimios A.; La Noce, Marcella

    2016-01-01

    Human dental pulp stem cells (hDPSCs), selected from the stromal-vascular fraction of dental pulp, are ecto-mesenchymal stem cells deriving from neural crests, successfully used in human bone tissue engineering. For their use in human therapy GMP procedures are required. For instance, the use of fetal bovine serum (FBS) is strongly discouraged in clinical practice due to its high risk of prions and other infections for human health. Alternatively, clinical grade sera have been suggested, including the New Zealand FBS (NZ-FBS). Therefore, the aim of this study was to evaluate the behavior of hDPSCs expanded in culture medium containing NZ-FBS. Since it was widely demonstrated hDPSCs display relevant capabilities to differentiate into osteogenic and angiogenic lineages, we performed a comparative study to assess if these features are also retained by cultivating the cells with a safer serum never tested on this cell line. hDPSCs were grown using NZ-FBS and conventional (C-FBS) for 7, 14, and 21 days, in both 2D and 3D cultures. Growth curves, expression of bone-related markers, calcification and angiogenesis were evaluated. NZ-FBS induced significant cell growth with respect to C-FBS and promoted an earlier increase expression of osteogenic markers, in particular of those involved in the formation of mineralized matrix (BSP and OPN) within 14 days. In addition, hDPSCs cultured in presence of NZ-FBS were found to produce higher mRNA levels of the angiogenic factors, such as VEGF and PDGFA. Taken together, our results highlight that hDPSCs proliferate, enhance their osteogenic commitment and increase angiogenic factors in NZ-FBS containing medium. These features have also been found when hDPSC were seeded on the clinical-grade collagen I scaffold (Bio-Gide®), leading to the conclusion that for human therapy some procedures and above all the use of GMP-approved materials have no negative impact. PMID:27594842

  11. NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression.

    PubMed

    Spina, Anna; Montella, Roberta; Liccardo, Davide; De Rosa, Alfredo; Laino, Luigi; Mitsiadis, Thimios A; La Noce, Marcella

    2016-01-01

    Human dental pulp stem cells (hDPSCs), selected from the stromal-vascular fraction of dental pulp, are ecto-mesenchymal stem cells deriving from neural crests, successfully used in human bone tissue engineering. For their use in human therapy GMP procedures are required. For instance, the use of fetal bovine serum (FBS) is strongly discouraged in clinical practice due to its high risk of prions and other infections for human health. Alternatively, clinical grade sera have been suggested, including the New Zealand FBS (NZ-FBS). Therefore, the aim of this study was to evaluate the behavior of hDPSCs expanded in culture medium containing NZ-FBS. Since it was widely demonstrated hDPSCs display relevant capabilities to differentiate into osteogenic and angiogenic lineages, we performed a comparative study to assess if these features are also retained by cultivating the cells with a safer serum never tested on this cell line. hDPSCs were grown using NZ-FBS and conventional (C-FBS) for 7, 14, and 21 days, in both 2D and 3D cultures. Growth curves, expression of bone-related markers, calcification and angiogenesis were evaluated. NZ-FBS induced significant cell growth with respect to C-FBS and promoted an earlier increase expression of osteogenic markers, in particular of those involved in the formation of mineralized matrix (BSP and OPN) within 14 days. In addition, hDPSCs cultured in presence of NZ-FBS were found to produce higher mRNA levels of the angiogenic factors, such as VEGF and PDGFA. Taken together, our results highlight that hDPSCs proliferate, enhance their osteogenic commitment and increase angiogenic factors in NZ-FBS containing medium. These features have also been found when hDPSC were seeded on the clinical-grade collagen I scaffold (Bio-Gide®), leading to the conclusion that for human therapy some procedures and above all the use of GMP-approved materials have no negative impact. PMID:27594842

  12. NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression

    PubMed Central

    Spina, Anna; Montella, Roberta; Liccardo, Davide; De Rosa, Alfredo; Laino, Luigi; Mitsiadis, Thimios A.; La Noce, Marcella

    2016-01-01

    Human dental pulp stem cells (hDPSCs), selected from the stromal-vascular fraction of dental pulp, are ecto-mesenchymal stem cells deriving from neural crests, successfully used in human bone tissue engineering. For their use in human therapy GMP procedures are required. For instance, the use of fetal bovine serum (FBS) is strongly discouraged in clinical practice due to its high risk of prions and other infections for human health. Alternatively, clinical grade sera have been suggested, including the New Zealand FBS (NZ-FBS). Therefore, the aim of this study was to evaluate the behavior of hDPSCs expanded in culture medium containing NZ-FBS. Since it was widely demonstrated hDPSCs display relevant capabilities to differentiate into osteogenic and angiogenic lineages, we performed a comparative study to assess if these features are also retained by cultivating the cells with a safer serum never tested on this cell line. hDPSCs were grown using NZ-FBS and conventional (C-FBS) for 7, 14, and 21 days, in both 2D and 3D cultures. Growth curves, expression of bone-related markers, calcification and angiogenesis were evaluated. NZ-FBS induced significant cell growth with respect to C-FBS and promoted an earlier increase expression of osteogenic markers, in particular of those involved in the formation of mineralized matrix (BSP and OPN) within 14 days. In addition, hDPSCs cultured in presence of NZ-FBS were found to produce higher mRNA levels of the angiogenic factors, such as VEGF and PDGFA. Taken together, our results highlight that hDPSCs proliferate, enhance their osteogenic commitment and increase angiogenic factors in NZ-FBS containing medium. These features have also been found when hDPSC were seeded on the clinical-grade collagen I scaffold (Bio-Gide®), leading to the conclusion that for human therapy some procedures and above all the use of GMP-approved materials have no negative impact.

  13. The expression of a bean PGIP in transgenic wheat confers increased resistance to the fungal pathogen Bipolaris sorokiniana.

    PubMed

    Janni, Michela; Sella, Luca; Favaron, Francesco; Blechl, Ann E; De Lorenzo, Giulia; D'Ovidio, Renato

    2008-02-01

    A possible strategy to control plant pathogens is the improvement of natural plant defense mechanisms against the tools that pathogens commonly use to penetrate and colonize the host tissue. One of these mechanisms is represented by the host plant's ability to inhibit the pathogen's capacity to degrade plant cell wall polysaccharides. Polygalacturonase-inhibiting proteins (PGIP) are plant defense cell wall glycoproteins that inhibit the activity of fungal endopolygalacturonases (endo-PGs). To assess the effectiveness of these proteins in protecting wheat from fungal pathogens, we produced a number of transgenic wheat lines expressing a bean PGIP (PvPGIP2) having a wide spectrum of specificities against fungal PGs. Three independent transgenic lines were characterized in detail, including determination of the levels of PvPGIP2 accumulation and its subcellular localization and inhibitory activity. Results show that the transgene-encoded protein is correctly secreted into the apoplast, maintains its characteristic recognition specificities, and endows the transgenic wheat with new PG recognition capabilities. As a consequence, transgenic wheat tissue showed increased resistance to digestion by the PG of Fusarium moniliforme. These new properties also were confirmed at the plant level during interactions with the fungal pathogen Bipolaris sorokiniana. All three lines showed significant reductions in symptom progression (46 to 50%) through the leaves following infection with this pathogen. Our results illustrate the feasibility of improving wheat's defenses against pathogens by expression of proteins with new capabilities to counteract those produced by the pathogens.

  14. DNA damage promotes Herpes Simplex Virus-1 protein expression in a neuroblastoma cell line

    PubMed Central

    Volcy, Ketna; Fraser, Nigel W.

    2013-01-01

    Although the induction of the cellular DNA damage response by Herpes simplex virus-1 (HSV-1) infection of epithelial cells in tissue culture promotes productive infection, there has been no experimental observation of the effect of the cellular DNA damage response on HSV-1 infection in vivo or in neuronal derived cell lines in tissue culture. Thus, it has been speculated that the lack of cellular DNA damage induction during infection of neurons may promote latency in these cells. This work examines the profile of HSV-1 promoter induction and protein expression, in the absence or presence of infection; using cellular DNA damage inducing topoisomerase inhibitors (Camptothecin and Etoposide) on a neuroblastoma cell line (C1300) in which HSV-1 infection fails to induce the DNA damage response. In the absence of infection, a plasmid expressing the immediate early ICP0 promoter was the most induced by the DNA damage drug treatments compared to the early (RR) and late (VP16) gene promoters. Similarly, drug treatment of C1300 cells infected with HSV-1 virus showed enhanced protein expression for ICP0, but not ICP4 and VP16 proteins. However, when the cells were infected with a HSV-1 virus defective in the immediate early gene trans-activator VP16 (in814) and treated with the DNA damaging drugs, there was enhanced expression of immediate early and late HSV-1 proteins. Although, viral infection of the neuroblastoma cell alone did not induce DNA damage, cellular DNA damage induced by drug treatments facilitated viral promoter induction and viral protein expression. This implicates a mechanism by which HSV-1 viral genes in a quiescent or latent state may become induced by cellular DNA damage in neuronal cells to facilitate productive infection. PMID:23354549

  15. Bestrophin expression and function in the human pancreatic duct cell line, CFPAC-1

    PubMed Central

    Marsey, Laura L; Winpenny, John P

    2009-01-01

    Pancreatic duct epithelial cells (PDECs) have been shown to express calcium activated chloride channels (CaCCs) and there is evidence for their involvement in fluid secretion from these cells. The molecular identity of the CaCC in PDECs remains unknown. Recently, the bestrophin family of proteins have been proposed as a potential molecular candidate for CaCCs. Expression of bestrophins is strongly correlated with the function of CaCCs in a variety of tissues. In the present study, the expression of bestrophins has been investigated in the cystic fibrosis pancreatic duct cell line, CFPAC-1. Iodide efflux analysis was used to characterise native CaCCs in CFPAC-1 cell monolayers. Efflux was induced with the addition of UTP (100 μm, 10.2 ± 1.5 nmol min−1), which was blocked by the chloride channel blockers niflumic acid (81%) and DIDS (90%). The UTP-stimulated iodide efflux was shown to be Ca2+ dependent and cAMP independent. RT-PCR analysis of RNA isolated from CFPAC-1 cells demonstrated positive identification of all four human bestrophin mRNAs. Western blot of CFPAC-1 cell protein isolates with antibodies specific to human bestrophin 1 (hBest1) showed that hBest1 protein was expressed in this cell line. HBest1 was present on the cell surface, demonstrated using biotinylation and confocal imaging, as well as in the cytoplasm. SiRNA-mediated silencing of hBest1 in CFPAC-1 cells reduced the UTP-stimulated iodide efflux by around 40%. This study provides evidence that the bestrophins are expressed in pancreatic duct cells and, more specifically, that hBest1 plays a role in the CaCCs found in these cells. PMID:19237432

  16. A minimal cytomegalovirus intron A variant can improve transgene expression in different mammalian cell lines.

    PubMed

    Quilici, L S; Silva-Pereira, I; Andrade, A C; Albuquerque, F C; Brigido, M M; Maranhão, A Q

    2013-01-01

    The expression enhancement by cytomegalovirus promoter and different intron A (IA) variants were evaluated in CHO-K1, HepG2, HEK-293 and COS-7 cells by assessing the levels of luciferase activity. This data along with mRNA levels measurement indicated that the construct harboring an IA variant with a 200-nucleotide deletion (Δ200) had the greatest impact on increasing luciferase expression among all constructs evaluated. Based on these results, we redesigned pCMV-IA variants and cloned them into plasmids expressing a humanized antibody. These plasmids were then used to transfect CHO-K1 cells. Production of the antibody was not augmented with the Δ200 promoter variant. The 600-nucleotide deletion (Δ600) and whole IA promoter variants expressed similar levels of the recombinant protein. These data indicate that the IA-based enhanced expression of transgenes depends on a small region within the intron.

  17. Phenolic compounds and expression of 4CL genes in silver birch clones and Pt4CL1a lines.

    PubMed

    Sutela, Suvi; Hahl, Terhi; Tiimonen, Heidi; Aronen, Tuija; Ylioja, Tiina; Laakso, Tapio; Saranpää, Pekka; Chiang, Vincent; Julkunen-Tiitto, Riitta; Häggman, Hely

    2014-01-01

    A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins.

  18. Phenolic Compounds and Expression of 4CL Genes in Silver Birch Clones and Pt4CL1a Lines

    PubMed Central

    Sutela, Suvi; Hahl, Terhi; Tiimonen, Heidi; Aronen, Tuija; Ylioja, Tiina; Laakso, Tapio; Saranpää, Pekka; Chiang, Vincent; Julkunen-Tiitto, Riitta; Häggman, Hely

    2014-01-01

    A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins. PMID:25502441

  19. Phenolic compounds and expression of 4CL genes in silver birch clones and Pt4CL1a lines.

    PubMed

    Sutela, Suvi; Hahl, Terhi; Tiimonen, Heidi; Aronen, Tuija; Ylioja, Tiina; Laakso, Tapio; Saranpää, Pekka; Chiang, Vincent; Julkunen-Tiitto, Riitta; Häggman, Hely

    2014-01-01

    A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins. PMID:25502441

  20. Epidermal growth factor increases LRF/Pokemon expression in human prostate cancer cells.

    PubMed

    Aggarwal, Himanshu; Aggarwal, Anshu; Agrawal, Devendra K

    2011-10-01

    Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis. PMID:21640721

  1. Epidermal growth factor increases LRF/Pokemon expression in human prostate cancer cells.

    PubMed

    Aggarwal, Himanshu; Aggarwal, Anshu; Agrawal, Devendra K

    2011-10-01

    Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis.

  2. Increasing The Genetic Admixture of Available Lines of Human Pluripotent Stem Cells

    PubMed Central

    Tofoli, Fabiano A.; Dasso, Maximiliano; Morato-Marques, Mariana; Nunes, Kelly; Pereira, Lucas Assis; da Silva, Giselle Siqueira; Fonseca, Simone A. S.; Costas, Roberta Montero; Santos, Hadassa Campos; da Costa Pereira, Alexandre; Lotufo, Paulo A.; Bensenor, Isabela M.; Meyer, Diogo; Pereira, Lygia Veiga

    2016-01-01

    Human pluripotent stem cells (hPSCs) may significantly improve drug development pipeline, serving as an in vitro system for the identification of novel leads, and for testing drug toxicity. Furthermore, these cells may be used to address the issue of differential drug response, a phenomenon greatly influenced by genetic factors. This application depends on the availability of hPSC lines from populations with diverse ancestries. So far, it has been reported that most lines of hPSCs derived worldwide are of European or East Asian ancestries. We have established 23 lines of hPSCs from Brazilian individuals, and we report the analysis of their genomic ancestry. We show that embryo-derived PSCs are mostly of European descent, while induced PSCs derived from participants of a national-wide Brazilian cohort study present high levels of admixed European, African and Native American genomic ancestry. Additionally, we use high density SNP data and estimate local ancestries, particularly those of CYP genes loci. Such information will be of key importance when interpreting variation among cell lines with respect to cellular phenotypes of interest. The availability of genetically admixed lines of hPSCs will be of relevance when setting up future in vitro studies of drug response. PMID:27708369

  3. NMDA receptors are expressed in human ovarian cancer tissues and human ovarian cancer cell lines

    PubMed Central

    North, William G; Liu, Fuli; Tian, Ruiyang; Abbasi, Hamza; Akerman, Bonnie

    2015-01-01

    We have earlier demonstrated that breast cancer and small-cell lung cancer express functional NMDA receptors that can be targeted to promote cancer cell death. Human ovarian cancer tissues and human ovarian cancer cell lines (SKOV3, A2008, and A2780) have now been shown to also express NMDA-receptor subunit 1 (GluN1) and subunit 2B (GluN2B). Seventeen ovarian cancers in two arrays were screened by immunohistochemistry using polyclonal antibodies that recognize an extracellular moiety on GluN1 and on GluN2B. These specimens comprised malignant tissue with pathology diagnoses of serous papillary cystadenocarcinoma, endometrioid adenocarcinoma, and clear-cell carcinoma. Additionally, archival tissues defined as ovarian adenocarcinoma from ten patients treated at this institute were also evaluated. All of the cancerous tissues demonstrated positive staining patterns with the NMDA-receptor antibodies, while no staining was found for tumor-adjacent normal tissues or sections of normal ovarian tissue. Human ovarian adenocarcinoma cell lines (A2008, A2780, SKOV3) were demonstrated to express GluN1 by Western blotting, but displayed different levels of expression. Through immunocytochemistry utilizing GluN1 antibodies and imaging using a confocal microscope, we were able to demonstrate that GluN1 protein is expressed on the surface of these cells. In addition to these findings, GluN2B protein was demonstrated to be expressed using polyclonal antibodies against this protein. Treatment of all ovarian cell lines with antibodies against GluN1 was found to result in decreased cell viability (P<0.001), with decreases to 10%–25% that of untreated cells. Treatment of control HEK293 cells with various dilutions of GluN1 antibodies had no effect on cell viability. The GluN1 antagonist MK-801 (dizocilpine maleate) and the GluN2B antagonist ifenprodil, like antibodies, dramatically decreased the viability of A2780 ovarian tumor cells (P<0.01). Treatment of A2780 tumor xenografts with

  4. NMDA receptors are expressed in human ovarian cancer tissues and human ovarian cancer cell lines.

    PubMed

    North, William G; Liu, Fuli; Tian, Ruiyang; Abbasi, Hamza; Akerman, Bonnie

    2015-01-01

    We have earlier demonstrated that breast cancer and small-cell lung cancer express functional NMDA receptors that can be targeted to promote cancer cell death. Human ovarian cancer tissues and human ovarian cancer cell lines (SKOV3, A2008, and A2780) have now been shown to also express NMDA-receptor subunit 1 (GluN1) and subunit 2B (GluN2B). Seventeen ovarian cancers in two arrays were screened by immunohistochemistry using polyclonal antibodies that recognize an extracellular moiety on GluN1 and on GluN2B. These specimens comprised malignant tissue with pathology diagnoses of serous papillary cystadenocarcinoma, endometrioid adenocarcinoma, and clear-cell carcinoma. Additionally, archival tissues defined as ovarian adenocarcinoma from ten patients treated at this institute were also evaluated. All of the cancerous tissues demonstrated positive staining patterns with the NMDA-receptor antibodies, while no staining was found for tumor-adjacent normal tissues or sections of normal ovarian tissue. Human ovarian adenocarcinoma cell lines (A2008, A2780, SKOV3) were demonstrated to express GluN1 by Western blotting, but displayed different levels of expression. Through immunocytochemistry utilizing GluN1 antibodies and imaging using a confocal microscope, we were able to demonstrate that GluN1 protein is expressed on the surface of these cells. In addition to these findings, GluN2B protein was demonstrated to be expressed using polyclonal antibodies against this protein. Treatment of all ovarian cell lines with antibodies against GluN1 was found to result in decreased cell viability (P<0.001), with decreases to 10%-25% that of untreated cells. Treatment of control HEK293 cells with various dilutions of GluN1 antibodies had no effect on cell viability. The GluN1 antagonist MK-801 (dizocilpine maleate) and the GluN2B antagonist ifenprodil, like antibodies, dramatically decreased the viability of A2780 ovarian tumor cells (P<0.01). Treatment of A2780 tumor xenografts with

  5. Increase of hepatic fat accumulation by liver specific expression of Hepatitis B virus X protein in zebrafish.

    PubMed

    Shieh, Yun-Sheng; Chang, Yin-Shan; Hong, Jiann-Ruey; Chen, Li-Je; Jou, Luen-Kuang; Hsu, Chia-Chun; Her, Guor Mour

    2010-07-01

    The pathogenesis of fatty liver disease remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis in zebrafish by liver specific expression of Hepatitis B virus X protein (HBx). Transgenic zebrafish lines, GBXs, which selectively express the GBx transgene (GFP-fused HBx gene) in liver, were established. GBX Liver phenotypes were evaluated by histopathology and molecular analysis of fatty acid (FA) metabolism-related genes expression. Most GBXs (66-81%) displayed obvious emaciation starting at 4 months old. Over 99% of the emaciated GBXs developed hepatic steatosis or steatohepatitis, which in turn led to liver hypoplasia. The liver histology of GBXs displayed steatosis, lobular inflammation, and balloon degeneration, similar to non-alcoholic steatohepatitis (NASH). Oil red O stain detected the accumulation of fatty droplets in GBXs. RT-PCR and Q-rt-PCR analysis revealed that GBx induced hepatic steatosis had significant increases in the expression of lipogenic genes, C/EBP-alpha, SREBP1, ChREBP and PPAR-gamma, which then activate key enzymes of the de novo FA synthesis, ACC1, FAS, SCD1, AGAPT, PAP and DGAT2. In addition, the steatohepatitic GBX liver progressed to liver degeneration and exhibited significant differential gene expression in apoptosis and stress. The GBX models exhibited both the genetic and functional factors involved in lipid accumulation and steatosis-associated liver injury. In addition, GBXs with transmissible NASH-like phenotypes provide a promising model for studying liver disease. PMID:20416398

  6. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages.

    PubMed

    Kim, Mi Jin; Nepal, Saroj; Lee, Eung-Seok; Jeong, Tae Cheon; Kim, Sang-Hyun; Park, Pil-Hoon

    2013-11-15

    Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages. PMID:23978445

  7. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages.

    PubMed

    Kim, Mi Jin; Nepal, Saroj; Lee, Eung-Seok; Jeong, Tae Cheon; Kim, Sang-Hyun; Park, Pil-Hoon

    2013-11-15

    Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages.

  8. Agronomic nitrogen-use efficiency of rice can be increased by driving OsNRT2.1 expression with the OsNAR2.1 promoter.

    PubMed

    Chen, Jingguang; Zhang, Yong; Tan, Yawen; Zhang, Min; Zhu, Longlong; Xu, Guohua; Fan, Xiaorong

    2016-08-01

    The importance of the nitrate (NO3-) transporter for yield and nitrogen-use efficiency (NUE) in rice was previously demonstrated using map-based cloning. In this study, we enhanced the expression of the OsNRT2.1 gene, which encodes a high-affinity NO3- transporter, using a ubiquitin (Ubi) promoter and the NO3--inducible promoter of the OsNAR2.1 gene to drive OsNRT2.1 expression in transgenic rice plants. Transgenic lines expressing pUbi:OsNRT2.1 or pOsNAR2.1:OsNRT2.1 constructs exhibited the increased total biomass including yields of approximately 21% and 38% compared with wild-type (WT) plants. The agricultural NUE (ANUE) of the pUbi:OsNRT2.1 lines decreased to 83% of that of the WT plants, while the ANUE of the pOsNAR2.1:OsNRT2.1 lines increased to 128% of that of the WT plants. The dry matter transfer into grain decreased by 68% in the pUbi:OsNRT2.1 lines and increased by 46% in the pOsNAR2.1:OsNRT2.1 lines relative to the WT. The expression of OsNRT2.1 in shoot and grain showed that Ubi enhanced OsNRT2.1 expression by 7.5-fold averagely and OsNAR2.1 promoters increased by about 80% higher than the WT. Interestingly, we found that the OsNAR2.1 was expressed higher in all the organs of pUbi:OsNRT2.1 lines; however, for pOsNAR2.1:OsNRT2.1 lines, OsNAR2.1 expression was only increased in root, leaf sheaths and internodes. We show that increased expression of OsNRT2.1, especially driven by OsNAR2.1 promoter, can improve the yield and NUE in rice. PMID:26826052

  9. Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer's disease model cell line

    SciTech Connect

    Sung, Hye Youn; Choi, Eun Nam; Ahn Jo, Sangmee; Oh, Seikwan; Ahn, Jung-Hyuck

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Genome-wide DNA methylation pattern in Alzheimer's disease model cell line. Black-Right-Pointing-Pointer Integrated analysis of CpG methylation and mRNA expression profiles. Black-Right-Pointing-Pointer Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. Black-Right-Pointing-Pointer The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterations in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2 Prime -deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may

  10. A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines

    PubMed Central

    Boegel, Sebastian; Löwer, Martin; Bukur, Thomas; Sahin, Ugur; Castle, John C

    2014-01-01

    Cancer cell lines are a tremendous resource for cancer biology and therapy development. These multipurpose tools are commonly used to examine the genetic origin of cancers, to identify potential novel tumor targets, such as tumor antigens for vaccine devel­opment, and utilized to screen potential therapies in preclinical studies. Mutations, gene expression, and drug sensitivity have been determined for many cell lines using next-generation sequencing (NGS). However, the human leukocyte antigen (HLA) type and HLA expression of tumor cell lines, characterizations necessary for the development of cancer vaccines, have remained largely incomplete and, such information, when available, has been distributed in many publications. Here, we determine the 4-digit HLA type and HLA expression of 167 cancer and 10 non-cancer cell lines from publically available RNA-Seq data. We use standard NGS RNA-Seq short reads from “whole transcriptome” sequencing, map reads to known HLA types, and statistically determine HLA type, heterozygosity, and expression. First, we present previously unreported HLA Class I and II genotypes. Second, we determine HLA expression levels in each cancer cell line, providing insights into HLA downregulation and loss in cancer. Third, using these results, we provide a fundamental cell line “barcode” to track samples and prevent sample annotation swaps and contamination. Fourth, we integrate the cancer cell-line specific HLA types and HLA expression with available cell-line specific mutation information and existing HLA binding prediction algorithms to make a catalog of predicted antigenic mutations in each cell line. The compilation of our results are a fundamental resource for all researchers selecting specific cancer cell lines based on the HLA type and HLA expression, as well as for the development of immunotherapeutic tools for novel cancer treatment modalities. PMID:25960936

  11. Nitric oxide increases Wnt-induced secreted protein-1 (WISP-1/CCN4) expression and function in colitis.

    PubMed

    Wang, Hongying; Zhang, Rui; Wen, Shoubin; McCafferty, Donna-Marie; Beck, Paul L; MacNaughton, Wallace K

    2009-04-01

    Nitric oxide (NO) derived from the inducible NO synthase (iNOS) is an important and complex mediator of inflammation in the intestine. Wnt-inducible secreted protein (WISP)-1 (CCN4), a member of the connective tissue growth factor family, is involved in tissue repair. We sought to determine the relationship between iNOS and WISP-1 in colitis. By analyzing human colonic biopsy samples, we showed that the expression of mRNA for both iNOS and WISP-1 was significantly higher in ulcerative colitis samples compared with control tissue. The upregulation of WISP-1 was positively correlated with iNOS expression in two models of colitis, induced by intrarectal trinitrobenzenesulfonic acid (TNBS) or occurring spontaneously in IL-10 deficient mice. Loss of iNOS, studied using iNOS(-/-) mice in both TNBS-induced and IL-10(-/-) colitis models, significantly attenuated the colitis-related WISP-1 increase. In human colonic epithelial cell lines, the NO donor, DETA-NONOate, elevated WISP-1 mRNA and protein expression through a beta-catenin and CREB-dependent, but Wnt-1-independent, pathway. In addition, NO-induced WISP-1 directly induced secretion of soluble collagen in colonic fibroblast cells. NO increases WISP-1 expression both in vitro and in vivo, suggesting a new role for iNOS and NO in colitis.

  12. A somaclonal line SE7 of finger millet (Eleusine coracana) exhibits modified cytokinin homeostasis and increased grain yield

    PubMed Central

    Radchuk, Volodymyr

    2012-01-01

    The SE7 somaclonal line of finger millet (Eleusine coracana) achieved increased grain yield in field trials that apparently resulted from a higher number of inflorescences and seeds per plant, compared with the wild type. Levels of endogenous cytokinins, especially those of highly physiologically active iso-pentenyl adenine, were increased during early inflorescence development in SE7 plants. Transcript levels of cytokinin-degrading enzymes but not of a cytokinin-synthesizing enzyme were also decreased in young leaves, seedlings, and initiating inflorescences of SE7. These data suggest that attenuated degradation of cytokinins in SE7 inflorescences leads to higher cytokinin levels that stimulate meristem activity and result in production of more inflorescences. Gene expression was compared between SE7 and wild-type young inflorescences using the barley 12K cDNA array. The largest fraction of up-regulated genes in SE7 was related to transcription, translation, and cell proliferation, cell wall assembly/biosynthesis, and to growth regulation of young and meristematic tissues including floral formation. Other up-regulated genes were associated with protein and lipid degradation and mitochondrial energy production. Down-regulated genes were related to pathogen defence and stress response, primary metabolism, glycolysis, and the C:N balance. The results indicate a prolonged proliferation phase in SE7 young inflorescences characterized by up-regulated protein synthesis, cytokinesis, floral formation, and energy production. In contrast, wild-type inflorescences are similar to a more differentiated status characterized by regulated protein degradation, cell elongation, and defence/stress responses. It is concluded that attenuated degradation of cytokinins in SE7 inflorescences leads to higher cytokinin levels, which stimulate meristem activity, inflorescence formation, and seed set. PMID:22888132

  13. Supercooling Capacity Increases from Sea Level to Tree Line in the Hawaiian Tree Species Metrosideros polymorpha.

    PubMed

    Melcher; Cordell; Jones; Scowcroft; Niemczura; Giambelluca; Goldstein

    2000-05-01

    Population-specific differences in the freezing resistance of Metrosideros polymorpha leaves were studied along an elevational gradient from sea level to tree line (located at ca. 2500 m above sea level) on the east flank of the Mauna Loa volcano in Hawaii. In addition, we also studied 8-yr-old saplings grown in a common garden from seeds collected from the same field populations. Leaves of low-elevation field plants exhibited damage at -2 degrees C, before the onset of ice formation, which occurred at -5.7 degrees C. Leaves of high-elevation plants exhibited damage at ca. -8.5 degrees C, concurrent with ice formation in the leaf tissue, which is typical of plants that avoid freezing in their natural environment by supercooling. Nuclear magnetic resonance studies revealed that water molecules of both extra- and intracellular leaf water fractions from high-elevation plants had restricted mobility, which is consistent with their low water content and their high levels of osmotically active solutes. Decreased mobility of water molecules may delay ice nucleation and/or ice growth and may therefore enhance the ability of plant tissues to supercool. Leaf traits that correlated with specific differences in supercooling capacity were in part genetically determined and in part environmentally induced. Evidence indicated that lower apoplastic water content and smaller intercellular spaces were associated with the larger supercooling capacity of the plant's foliage at tree line. The irreversible tissue-damage temperature decreased by ca. 7 degrees C from sea level to tree line in leaves of field populations. However, this decrease appears to be only large enough to allow M. polymorpha trees to avoid leaf tissue damage from freezing up to a level of ca. 2500 m elevation, which is also the current tree line location on the east flank of Mauna Loa. The limited freezing resistance of M. polymorpha leaves may be partially responsible for the occurrence of tree line at a relatively

  14. Supercooling Capacity Increases from Sea Level to Tree Line in the Hawaiian Tree Species Metrosideros polymorpha.

    PubMed

    Melcher; Cordell; Jones; Scowcroft; Niemczura; Giambelluca; Goldstein

    2000-05-01

    Population-specific differences in the freezing resistance of Metrosideros polymorpha leaves were studied along an elevational gradient from sea level to tree line (located at ca. 2500 m above sea level) on the east flank of the Mauna Loa volcano in Hawaii. In addition, we also studied 8-yr-old saplings grown in a common garden from seeds collected from the same field populations. Leaves of low-elevation field plants exhibited damage at -2 degrees C, before the onset of ice formation, which occurred at -5.7 degrees C. Leaves of high-elevation plants exhibited damage at ca. -8.5 degrees C, concurrent with ice formation in the leaf tissue, which is typical of plants that avoid freezing in their natural environment by supercooling. Nuclear magnetic resonance studies revealed that water molecules of both extra- and intracellular leaf water fractions from high-elevation plants had restricted mobility, which is consistent with their low water content and their high levels of osmotically active solutes. Decreased mobility of water molecules may delay ice nucleation and/or ice growth and may therefore enhance the ability of plant tissues to supercool. Leaf traits that correlated with specific differences in supercooling capacity were in part genetically determined and in part environmentally induced. Evidence indicated that lower apoplastic water content and smaller intercellular spaces were associated with the larger supercooling capacity of the plant's foliage at tree line. The irreversible tissue-damage temperature decreased by ca. 7 degrees C from sea level to tree line in leaves of field populations. However, this decrease appears to be only large enough to allow M. polymorpha trees to avoid leaf tissue damage from freezing up to a level of ca. 2500 m elevation, which is also the current tree line location on the east flank of Mauna Loa. The limited freezing resistance of M. polymorpha leaves may be partially responsible for the occurrence of tree line at a relatively

  15. Gene expression analysis of tuberous sclerosis complex cortical tubers reveals increased expression of adhesion and inflammatory factors

    PubMed Central

    Boer, Karin; Crino, Peter B.; Gorter, Jan A.; Nellist, Mark; Jansen, Floor E.; Spliet, Wim G.M.; van Rijen, Peter C.; Wittink, Floyd R.A.; Breit, Timo M.; Troost, Dirk; Wadman, Wytse J.; Aronica, Eleonora

    2009-01-01

    Cortical tubers in patients with tuberous sclerosis complex are associated with disabling neurological manifestations, including intractable epilepsy. While these malformations are believed to result from the effects of TSC1 or TSC2 gene mutations, the molecular mechanisms leading to tuber formation, as well as the onset of seizures remain largely unknown. We used the Affymetrix Gene Chip platform to provide the first genome wide investigation of gene expression in surgically resected tubers, compared with histological normal perituberal tissue from the same patients or autopsy control tissue. We identified 2501 differentially expressed genes in cortical tubers compared with autopsy controls. Expression of genes associated with cell adhesion e.g., VCAM1, integrins and CD44, or with the inflammatory response, including complement factors, serpinA3, CCL2 and several cytokines, was increased in cortical tubers, whereas genes related to synaptic transmission e.g., the glial glutamate transporter GLT-1, and voltage-gated channel activity, exhibited lower expression. Gene expression in perituberal cortex was distinct from autopsy control cortex suggesting that even in the absence of tissue pathology the transcriptome is altered in TSC. Changes in gene expression yield insights into new candidate genes that may contribute to tuber formation or seizure onset, representing new targets for potential therapeutic development. PMID:19912235

  16. Overexpression of several Arabidopsis histone genes increases agrobacterium-mediated transformation and transgene expression in plants.

    PubMed

    Tenea, Gabriela N; Spantzel, Joerg; Lee, Lan-Ying; Zhu, Yanmin; Lin, Kui; Johnson, Susan J; Gelvin, Stanton B

    2009-10-01

    The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double- or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation.

  17. Gene Expression Correlations in Human Cancer Cell Lines Define Molecular Interaction Networks for Epithelial Phenotype

    PubMed Central

    Kohn, Kurt W.; Zeeberg, Barry M.; Reinhold, William C.; Pommier, Yves

    2014-01-01

    Using gene expression data to enhance our knowledge of control networks relevant to cancer biology and therapy is a challenging but urgent task. Based on the premise that genes that are expressed together in a variety of cell types are likely to functions together, we derived mutually correlated genes that function together in various processes in epithelial-like tumor cells. Expression-correlated genes were derived from data for the NCI-60 human tumor cell lines, as well as data from the Broad Institute’s CCLE cell lines. NCI-60 cell lines that selectively expressed a mutually correlated subset of tight junction genes served as a signature for epithelial-like cancer cells. Those signature cell lines served as a seed to derive other correlated genes, many of which had various other epithelial-related functions. Literature survey yielded molecular interaction and function information about those genes, from which molecular interaction maps were assembled. Many of the genes had epithelial functions unrelated to tight junctions, demonstrating that new function categories were elicited. The most highly correlated genes were implicated in the following epithelial functions: interactions at tight junctions (CLDN7, CLDN4, CLDN3, MARVELD3, MARVELD2, TJP3, CGN, CRB3, LLGL2, EPCAM, LNX1); interactions at adherens junctions (CDH1, ADAP1, CAMSAP3); interactions at desmosomes (PPL, PKP3, JUP); transcription regulation of cell-cell junction complexes (GRHL1 and 2); epithelial RNA splicing regulators (ESRP1 and 2); epithelial vesicle traffic (RAB25, EPN3, GRHL2, EHF, ADAP1, MYO5B); epithelial Ca(+2) signaling (ATP2C2, S100A14, BSPRY); terminal differentiation of epithelial cells (OVOL1 and 2, ST14, PRSS8, SPINT1 and 2); maintenance of apico-basal polarity (RAB25, LLGL2, EPN3). The findings provide a foundation for future studies to elucidate the functions of regulatory networks specific to epithelial-like cancer cells and to probe for anti-cancer drug targets. PMID:24940735

  18. Increased expression of long intergenic non-coding RNA LINC00152 in gastric cancer and its clinical significance.

    PubMed

    Pang, Qianqian; Ge, Jiaxin; Shao, Yongfu; Sun, Weiliang; Song, Haojun; Xia, Tian; Xiao, Bingxiu; Guo, Junming

    2014-06-01

    It has been known that differential expression of long non-coding RNA (lncRNA) plays critical roles in carcinogenesis. However, the significance of lncRNA, especially long intergenic ncRNA (lincRNA, the main type of lncRNA family), in the diagnosis of gastric cancer is largely unknown. The aim of this study was to determine the expression level of LINC00152, a newfound lincRNA, in gastric carcinoma and its clinical association. The expression of LINC00152 in 71 pairs of tumorous and adjacent normal tissues from patients with gastric cancer was detected by quantitative real-time reverse transcription-polymerase chain reaction. And then, the potential associations between its level in gastric cancer tissue and the clinicopathological features were analyzed. Finally, a receiver operating characteristic (ROC) curve was constructed for differentiating patients with gastric cancer from patients with benign gastric diseases. The results showed that the expression level of LINC00152 in gastric carcinoma was significantly increased, compared with matched normal tissue (P=0.045) and normal mucosa from health control (P=0.004), respectively. Levels of LINC00152 in gastric cancer cell lines, BGC-823, MGC-803, and SGC-7901, were significantly higher than those in human normal gastric epithelial cell line GES-1. In addition, high expression of LINC00152 was correlated with invasion (P=0.042). LINC00152 levels in gastric juice from patients with gastric cancer were further found significantly higher than those from normal controls (P=0.002). Moreover, the area under the ROC curve (AUC) was up to 0.645 (95 % CI=0.559-0.740, P=0.003). This study highlights that lincRNA LINC00152 might be a novel biomarker for predicting gastric cancer.

  19. Identification of the thiamin pyrophosphokinase gene in rainbow trout: characteristic structure and expression of seven splice variants in tissues and cell lines and during embryo development

    USGS Publications Warehouse

    Yuge, Shinya; Richter, Catherine A.; Wright-Osment, Maureen K.; Nicks, Diane; Saloka, Stephanie K.; Tillitt, Donald E.; Li, Weiming

    2012-01-01

    Thiamin pyrophosphokinase (TPK) converts thiamin to its active form, thiamin diphosphate. In humans, TPK expression is down-regulated in some thiamin deficiency related syndrome, and enhanced during pregnancy. Rainbow trout are also vulnerable to thiamin deficiency in wild life and are useful models for thiamin metabolism research. We identified the tpk gene transcript including seven splice variants in the rainbow trout. Almost all cell lines and tissues examined showed co-expression of several tpk splice variants including a potentially major one at both mRNA and protein levels. However, relative to other tissues, the longest variant mRNA expression was predominant in the ovary and abundant in embryos. During embryogenesis, total tpk transcripts increased abruptly in early development, and decreased to about half of the peak shortly after hatching. In rainbow trout, the tpk transcript complex is ubiquitously expressed for all tissues and cells examined, and its increase in expression could be important in the early-middle embryonic stages. Moreover, decimated tpk expression in a hepatoma cell line relative to hepatic and gonadal cell lines appears to be consistent with previously reported down-regulation of thiamin metabolism in cancer.

  20. Increased aquaglyceroporin 9 expression disrupts arsenic resistance in human lung cancer cells.

    PubMed

    Miao, Zhi-Feng; Chang, Eddy Essen; Tsai, Feng-Yuan; Yeh, Szu-Ching; Wu, Chia-Fang; Wu, Kuen-Yuh; Wang, Chien-Jen; Tsou, Tsui-Chun

    2009-03-01

    Resistance to chemotherapy is one of the major problems in treatment responses of lung cancer. This study explored the mechanism underlying the arsenic resistance of lung cancer. Four lung cancer cells with different proliferation activity were characterized for cytotoxicity, arsenic influx/efflux, and arsenic effects on intracellular glutathione and 8-hydroxy-2'-deoxyguanosine (8-OHdG) production. Our data revealed that relative proliferation potency of these cells was H1299>A549>CL3>H1355. Moreover, A549, H1299, and H1355 were markedly resistant to As(2)O(3) with IC50 approximately 100 microM, whereas CL3 was sensitive to As(2)O(3) with IC50 approximately 11.8 microM. After treatment with the respective As(2)O(3) at IC50, arsenic influx/efflux activity in CL3 was comparable to those in the other three arsenic-resistant cells. However, differences in glutathione levels and 8-OHdG production were also detected either before or after arsenic treatment, indicating that a certain degree of variation in anti-oxidative systems and/or 8-OHdG repair activity existed in these cell lines. By transfection of an aquaglyceroporin 9 (AQP9) gene, we showed that increased AQP9 expression significantly enhanced arsenic uptake and disrupted arsenic resistance of A549. The present study strongly suggests that membrane transporters responsible for arsenic uptake, such as AQP9, may play a critical role in development of arsenic resistance in human lung cancer cells.

  1. Metformin-induced inhibition of the mitochondrial respiratory chain increases FGF21 expression via ATF4 activation

    SciTech Connect

    Kim, Kook Hwan; Jeong, Yeon Taek; Kim, Seong Hun; Jung, Hye Seung; Park, Kyong Soo; Lee, Hae-Youn; Lee, Myung-Shik

    2013-10-11

    Highlights: •Metformin induces FGF21 expression in an AMPK independent manner. •Metformin enhances FGF21 expression by inhibiting mitochondrial complex I activity. •The PERK-eIF2α-ATF4 axis is required for metformin-induced FGF21 expression. •Metformin activates the ATF4-FGF21 axis in the liver of mouse. •Metformin increases serum FGF21 level in diabetic human subjects. -- Abstract: Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exhibits anti-obesity and anti-diabetes effects. Because metformin is widely used as a glucose-lowering agent in patients with type 2 diabetes (T2D), we investigated whether metformin modulates FGF21 expression in cell lines, and in mice or human subjects. We found that metformin increased the expression and release of FGF21 in a diverse set of cell types, including rat hepatoma FaO, primary mouse hepatocytes, and mouse embryonic fibroblasts (MEFs). Intriguingly, AMP-activated protein kinase (AMPK) was dispensable for the induction of FGF21 by metformin. Mammalian target of rapamycin complex 1 (mTORC1) and peroxisome proliferator-activated receptor α (PPARα), which are additional targets of metformin, were not involved in metformin-induced FGF21 expression. Importantly, inhibition of mitochondrial complex I activity by metformin resulted in FGF21 induction through PKR-like ER kinase (PERK)-eukaryotic translation factor 2α (eIF2α)-activating transcription factor 4 (ATF4). We showed that metformin activated ATF4 and increased FGF21 expression in the livers of mice, which led to increased serum levels of FGF21. We also found that serum FGF21 level was increased in human subjects with T2D after metformin therapy for 6 months. In conclusion, our results indicate that metformin induced expression of FGF21 through an ATF4-dependent mechanism by inhibiting mitochondrial respiration independently of AMPK. Therefore, FGF21 induction by metformin might explain a portion of the beneficial metabolic effects of metformin.

  2. Expression of corticosteroid-binding globulin in human astrocytoma cell line.

    PubMed

    Pusch, Larissa; Wegmann, Sonja; Caldwell, Jack D; Jirikowski, Gustav F

    2009-06-01

    Glial tumor cells are known to be sensitive to glucocorticoids (GC) in vivo and in vitro. Here we studied the expression of corticosteroid-binding globulin (CBG) in the low-grade malignant human astrocytoma cell line 1321N1. CBG was observed in cytoplasm of most of these cells with immunocytochemistry. RT-PCR revealed the presence of the respective mRNA. Only scattered cells contained nuclear immunoreactivity for glucocorticoid receptor as visualized by double immunostaining. Immunoreactive CBG could be recovered from the supernatant of cultures that had been exposed to 10(-5) M cortisol. Our observations indicate the endogenous expression of CBG in 1321N1 cells which may occur independently from classical glucocorticoid receptor pathways. Cortisol seems to facilitate liberation of CBG in a paracrine manner, perhaps through membrane action of the steroid. Effects of adrenal steroids on proliferation and apoptosis of certain glial tumors may in part depend on these mechanisms.

  3. Increased expression of dermatopontin and its implications for testicular dysfunction in mice

    PubMed Central

    CAI, JUN; LIU, WEIJIA; HAO, JIE; CHEN, MAOXIN; LI, GANG

    2016-01-01

    An array of specific and non-specific molecules, which are expressed in the testis, have been demonstrated to be responsible for testicular function. Our previous study revealed that dermatopontin (DPT) is expressed in Sertoli cells of the testis, however, its roles in testicular function remains somewhat elusive. In the present study, CdCl2- and busulfan-induced testicular dysfunction models were used to investigate the implications of DPT expression for testicular function. The mRNA and protein expression levels of DPT were detected using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. A negative correlation was observed between testicular damage and the expression of DPT, which suggested that an increase in DPT expression may be a marker for testicular dysfunction. This result was corroborated by the finding that transgenic mice exhibiting Sertoli cell-specific overexpression of DPT exhibited damage to their testicular morphology. Additionally, DPT overexpression in the testis affected the expression levels of claudin-11 and zonula occludens-1, which indicated that DPT may affect testicular function by affecting the integrity of the blood-testis barrier (BTB). In conclusion, the present study provided evidence to suggest that DPT may be indicative of mouse testicular dysfunction, since increased expression may be associated with damage to the BTB. PMID:26861869

  4. Increased NY-ESO-1 expression and reduced infiltrating CD3+ T cells in cutaneous melanoma.

    PubMed

    Giavina-Bianchi, Mara; Giavina-Bianchi, Pedro; Sotto, Mirian Nacagami; Muzikansky, Alona; Kalil, Jorge; Festa-Neto, Cyro; Duncan, Lyn M

    2015-01-01

    NY-ESO-1 is a cancer-testis antigen aberrantly expressed in melanomas, which may serve as a robust and specific target in immunotherapy. NY-ESO-1 antigen expression, tumor features, and the immune profile of tumor infiltrating lymphocytes were assessed in primary cutaneous melanoma. NY-ESO-1 protein was detected in 20% of invasive melanomas (16/79), rarely in in situ melanoma (1/10) and not in benign nevi (0/20). Marked intratumoral heterogeneity of NY-ESO-1 protein expression was observed. NY-ESO-1 expression was associated with increased primary tumor thickness (P = 0.007) and inversely correlated with superficial spreading melanoma (P < 0.02). NY-ESO-1 expression was also associated with reduced numbers and density of CD3+ tumor infiltrating lymphocytes (P = 0.017). When NY-ESO-1 protein was expressed, CD3+ T cells were less diffusely infiltrating the tumor and were more often arranged in small clusters (P = 0.010) or as isolated cells (P = 0.002) than in large clusters of more than five lymphocytes. No correlation of NY-ESO-1 expression with gender, age, tumor site, ulceration, lymph node sentinel status, or survival was observed. NY-ESO-1 expression in melanoma was associated with tumor progression, including increased tumor thickness, and with reduced tumor infiltrating lymphocytes.

  5. Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

    PubMed

    Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

    2015-12-01

    Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese.

  6. Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

    PubMed

    Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

    2015-12-01

    Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese. PMID:26341925

  7. Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

    PubMed Central

    Hammer, Susanne C.; Becker, Annegret; Rateitschak, Katja; Mohr, Annika; Lüder Ripoli, Florenza; Hennecke, Silvia; Junginger, Johannes; Hewicker-Trautwein, Marion; Brenig, Bertram; Ngezahayo, Anaclet; Nolte, Ingo; Murua Escobar, Hugo

    2016-01-01

    Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies. PMID:27690019

  8. [Construction of Fat-1 eukaryotic expression vector of excision markers and the establishment of transgenic sheep cell lines].

    PubMed

    Lima, A; Zhu, Heping; Wang, Ruiyao; Yan, Tao; Su, Xiaohu; Li, Lu; Wang, Bingping; Na, Shunwendoule; Qi, Guichun; Zhou, Huanmin

    2016-02-01

    In order to establish marker-free transgenic cell lines, we cloned Fat-1 gene, attB and Loxp sequences by PCR. Then we inserted these sequences to pN1-EGFP vector and got pEGFP-N1-Fat-1 expression vector. PhiC31 integrase mRNA which was generated by in vitro transcription and a pEGFP-N1-Fat-1 expression vector co-electroporated into sheep fetal fibroblasts, and then we got transgenic cell lines expressing green fluorescence. Prokaryotic expression and purification of Cre recombinant protein was performed. Cre recombinant protein was transducted into stably-transfected cell colonies. We identified cell colonies by sequencing and established marker-free transgenic cell lines and eventually- established marker-free transgenic cell lines which were building more safely basic for producing Fat-1 transgenic animals. PMID:27382771

  9. [Construction of Fat-1 eukaryotic expression vector of excision markers and the establishment of transgenic sheep cell lines].

    PubMed

    Lima, A; Zhu, Heping; Wang, Ruiyao; Yan, Tao; Su, Xiaohu; Li, Lu; Wang, Bingping; Na, Shunwendoule; Qi, Guichun; Zhou, Huanmin

    2016-02-01

    In order to establish marker-free transgenic cell lines, we cloned Fat-1 gene, attB and Loxp sequences by PCR. Then we inserted these sequences to pN1-EGFP vector and got pEGFP-N1-Fat-1 expression vector. PhiC31 integrase mRNA which was generated by in vitro transcription and a pEGFP-N1-Fat-1 expression vector co-electroporated into sheep fetal fibroblasts, and then we got transgenic cell lines expressing green fluorescence. Prokaryotic expression and purification of Cre recombinant protein was performed. Cre recombinant protein was transducted into stably-transfected cell colonies. We identified cell colonies by sequencing and established marker-free transgenic cell lines and eventually- established marker-free transgenic cell lines which were building more safely basic for producing Fat-1 transgenic animals.

  10. Reactive oxygen species enhance TLR10 expression in the human monocytic cell line THP-1.

    PubMed

    Kim, Donghee; Kim, Yeon Ju; Koh, Hyun Sook; Jang, Tae Yang; Park, Hyo Eun; Kim, Jae Young

    2010-01-01

    We investigated TLR10 expression in human monocytes, THP-1 cells, cultured in hypoxia (3% O(2)). Levels of both TLR10 mRNA and protein in THP-1 cells cultured in hypoxia were significantly higher than those cultured in normoxia (20% O(2)). We examined intracellular reactive oxygen species (ROS) content in hypoxic cells, and TLR10 expression in cells treated with hydrogen peroxide (H(2)O(2)), to determine whether the increase in TLR10 expression observed with hypoxia was due to an increase in intracellular ROS levels. We found that the level of intracellular ROS in cells subject to hypoxia was significantly higher than in normoxia. Experiments with ROS synthesis inhibitors revealed that hypoxia induced ROS production is mainly due to NADPH oxidase activity. TLR10 mRNA expression was increased by treatment with H(2)O(2) at concentrations ranging from 50 to 250 μM. We screened the TLR10 promoter and found putative binding sites for transcription factors (TFs), such as NF-κB, NF-AT and AP-1. Next, we examined TF activities using a luciferase reporter assay. Activities of NF-κB, NF-AT and AP-1 in the cells treated with H(2)O(2) were significantly higher than in untreated cells. The experiment with TF inhibitors revealed that ROS-induced upregulation of TLR10 expression is mainly due to NF-κB activation. Overall, our results suggest that hypoxia or ROS increase TLR10 expression in human monocytes and the transcriptional activities of NF-κB are involved in this process. Therefore, it is suggested that ROS produced by various exogenous stimuli may play a crucial role in the regulation of expression and function of TLR10 as second messengers.

  11. Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line

    PubMed Central

    2011-01-01

    Background When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray. Results A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, http://www.ingenuity.com) revealed that 902 of 3876 differentially expressed genes were classified into a number of functional groups including cellular growth and proliferation, cell cycle, cellular movement, cancer, genetic disorders, and cell death. Also, the top 5 gene networks with intermolecular connections were identified. Bioinformatic analyses suggested that DF-1 cells were characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, altered cellular morphogenesis, and accelerated capacity for molecule transport. Key molecules for these functions include E2F1, BRCA1, SRC, CASP3, and the peroxidases. Conclusions The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells. PMID:22111699

  12. Gene expression patterns in near isogenic lines for wheat rust resistance gene lr34/yr18.

    PubMed

    Hulbert, S H; Bai, J; Fellers, J P; Pacheco, M G; Bowden, R L

    2007-09-01

    ABSTRACT The Lr34/Yr18 resistance gene provides durable, adult-plant, slow rusting resistance to leaf rust, yellow rust, and several other diseases of wheat. Flag leaves may exhibit spontaneous leaf tip necrosis and tips are more resistant than leaf bases. Despite the importance of this gene, the mechanism of resistance is unknown. Patterns of expression for 55,052 transcripts were examined by microarray analysis in mock-inoculated flag leaves of two pairs of wheat near isogenic lines for Lr34/Yr18 (Jupateco 73S/Jupateco 73R and Thatcher/Thatcher-Lr34). The Thatcher isolines were also examined for patterns of expression after inoculation with leaf rust. Mock-inoculated leaf tips of resistant plants showed up-regulation of 57 transcripts generally associated with ABA inducibility, osmotic stress, cold stress, and/or seed maturation. Several transcripts may be useful as expression markers for Lr34/Yr18. Five transcripts were also up-regulated in resistant leaf bases. The possible role of these transcripts in resistance is discussed. In mock-inoculated plants, pathogenesis-related (PR) proteins were not up-regulated in resistant flag leaves compared with that in susceptible flag leaves. In inoculated plants, the same set of PR proteins was up-regulated in both resistant and susceptible flag leaves. However, expression was often higher in resistant plants, suggesting a possible role for Lr34/Yr18 in priming of defense responses.

  13. Cardiomyocyte marker expression in a human lymphocyte cell line using mouse cardiomyocyte extract.

    PubMed

    Vojdani, Zahra; Tavakolinejad, Sima; Talaei-Khozani, Tahereh; Esmaeilpour, Tahereh; Rasooli, Manuchehr

    2011-03-01

    Cell transplantation shows potential for the treatment of cardiac diseases. Embryonic stem cells, cord blood and mesenchymal stem cells have been suggested as sources for transplantation therapy. Because of some technical limitations with the use of stem cells, transdifferentiation of fully differentiated cells is a potentially useful alternative. We investigated whether human peripheral blood cells could transdifferentiate into cardiomyocyte. Transdifferentiation was induced in a human B lymphocyte cell line (Raji). Cardiomyocyte extract was prepared from adult mouse cardiomyocytes. The cells were treated with 5-aza-2-deoxycytidine and trichostatin A, permeabilized with streptolysin O, and exposed to the mouse cardiomyocyte extract. They were cultured for 10 days, 3 weeks and 4 weeks. Cardiomyocyte markers were detected with immunohistochemistry and flow cytometry. Immunocytochemistry revealed that some cells expressed myosin heavy chain, α-actinin and cardiac troponin T after 3 and 4 weeks. Flow cytometry confirmed these data. In cells exposed to trichostatin A and 5-aza-2-deoxycytidine and permeabilized in the presence of the cardiomyocyte extract, troponin T expression was seen in 3.53% of the cells and 3.11% of them expressed α-actinin. After exposure to the cardiomyocyte extract, some permeabilized cells adhered to the plate loosely; however, the morphology did not change significantly, and they continued to show a rounded shape after 4 weeks. Our treated lymphocytes expressed cardiomyocyte markers. Our results suggest that lymphocytes may be useful in future research as a source of cells for reprogramming procedures.

  14. Increased Expression of the Na,K-ATPase alpha4 Isoform Enhances Sperm Motility in Transgenic Mice1

    PubMed Central

    Jimenez, Tamara; Sanchez, Gladis; McDermott, Jeffrey P.; Nguyen, Anh-Nguyet; Kumar, T. Rajendra; Blanco, Gustavo

    2010-01-01

    The Na,K-ATPase alpha4 (ATP1A4) isoform is specifically expressed in male germ cells and is highly prevalent in spermatozoa. Although selective inhibition of alpha4 activity with ouabain has been shown to affect sperm motility, a more direct analysis of the role of this isoform in sperm movement has not yet been demonstrated. To establish this, we engineered transgenic mice that express the rat alpha4 isoform fused to green fluorescent protein in male germ cells, under the control of the mouse protamine 1 promoter. We showed that the rat Atp1a4 transgene is expressed in mouse spermatozoa and that it is localized to the sperm flagellum. In agreement with increased expression of the alpha4 isoform, sperm from transgenic mice displayed higher alpha4-specific Na,K-ATPase activity and binding of fluorescently labeled ouabain than wild-type mice. In contrast, expression and activity of ATP1A1 (alpha1), the other Na,K-ATPase alpha isoform present in sperm, remained unchanged. Similar to wild-type mice, mice expressing the alpha4 transgene exhibited normal testis and sperm morphology and no differences in fertility. However, compared to wild-type mice, sperm from transgenic mice displayed plasma membrane hyperpolarization and higher total and progressive motility. Other parameters of motility also increased, including straight-line, curvilinear, and average path velocities and amplitude of lateral head displacement. In addition, sperm from the transgenic mice showed enhanced sperm hyperactive motility, but no changes in progesterone-induced acrosome reaction. Altogether, these results provide new genetic evidence for the role of the ATP1A4 isoform in sperm motility, under both noncapacitating and capacitating conditions. PMID:20826726

  15. Expression of Interferon Lambda 4 Is Associated with Reduced Proliferation and Increased Cell Death in Human Hepatic Cells

    PubMed Central

    Onabajo, Olusegun O.; Porter-Gill, Patricia; Paquin, Ashley; Rao, Nina; Liu, Luyang; Tang, Wei; Brand, Nathan

    2015-01-01

    Interferon lambda 4 (IFN-λ4) is a novel type-III interferon that can be generated only in individuals carrying a ΔG frame-shift allele of an exonic genetic variant (rs368234815-ΔG/TT). The rs368234815-ΔG allele is strongly associated with decreased clearance of hepatitis C virus (HCV) infection. Here, we further explored the biological function of IFN-λ4 expressed in human hepatic cells—a hepatoma cell line HepG2 and fresh primary human hepatocytes (PHHs). We performed live confocal imaging, cell death and proliferation assays, mRNA expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. Not only did we observe significant intracellular retention of IFN-λ4 but also detected secreted IFN-λ4 in the culture media of expressing cells. Secreted IFN-λ4 induced strong activation of the interferon-stimulated genes (ISGs) in IFN-λ4-expressing and surrounding cells in transwell assays. Specifically, in PHHs, secreted IFN-λ4 induced expression of the CXCL10 transcript and a corresponding pro-inflammatory chemokine, IP-10. In IFN-λ4-expressing HepG2 cells, we also observed decreased proliferation and increased cell death. All IFN-λ4-induced phenotypes—activation of ISGs, decreased proliferation, and increased cell death—could be inhibited by an anti-IFN-λ4-specific antibody. Our study offers new insights into biology of IFN-λ4 and its possible role in HCV clearance. PMID:26134097

  16. Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Frye, S. R.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

  17. Increased FasL expression correlates with apoptotic changes in granulocytes cultured with oxidized clozapine

    SciTech Connect

    Husain, Zaheed; Almeciga, Ingrid; Delgado, Julio C.; Clavijo, Olga P.; Castro, Januario E.; Belalcazar, Viviana; Pinto, Clara; Zuniga, Joaquin; Romero, Viviana; Yunis, Edmond J. . E-mail: edmond_yunis@dfci.harvard.edu

    2006-08-01

    Clozapine has been associated with a 1% incidence of agranulocytosis. The formation of an oxidized intermediate clozapine metabolite has been implicated in direct polymorphonuclear (PMN) toxicity. We utilized two separate systems to analyze the role of oxidized clozapine in inducing apoptosis in treated cells. Human PMN cells incubated with clozapine (0-10 {mu}M) in the presence of 0.1 mM H{sub 2}O{sub 2} demonstrated a progressive decrease of surface CD16 expression along with increased apoptosis. RT-PCR analysis showed decreased CD16 but increased FasL gene expression in clozapine-treated PMN cells. No change in constitutive Fas expression was observed in treated cells. In HL-60 cells induced to differentiate with retinoic acid (RA), a similar increase in FasL expression, but no associated changes in CD16 gene expression, was observed following clozapine treatments. Our results demonstrate increased FasL gene expression in oxidized clozapine-induced apoptotic neutrophils suggesting that apoptosis in granulocytes treated with clozapine involves Fas/FasL interaction that initiates a cascade of events leading to clozapine-induced agranulocytosis.

  18. A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2Apro: Effects on Cellular and Viral Gene Expression

    PubMed Central

    Barco, Angel; Feduchi, Elena; Carrasco, Luis

    2000-01-01

    A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2Apro) under the control of tetracycline has been obtained. Synthesis of 2Apro induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2Apro cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2Apro, prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2Apro still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2Apro. Moreover, synthesis of 2Apro in 2A7d cells complements the translational defect of a poliovirus 2Apro-defective variant. These results show that poliovirus 2Apro expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2Apro functions, to complement poliovirus 2Apro mutants, and to test antiviral compounds. PMID:10666269

  19. Expression and function of striatal nAChRs differ in the flinders sensitive (FSL) and resistant (FRL) rat lines.

    PubMed

    Auta, J; Lecca, D; Nelson, M; Guidotti, A; Overstreet, D H; Costa, E; Javaid, J I

    2000-10-01

    Rats of Flinders Sensitive (FSL) and Flinders Resistant lines (FRL) differ in their susceptibility to physiological and associated behavioral responses elicited by nicotine. In the present study, we measured dopamine (DA) content in striatal dialysates to investigate the sensitivity of FSL and FRL rats to nicotine delivered locally through a microdialysis probe placed in the striatum. We also measured the expression density of striatal high-affinity nicotinic acetylcholine receptors (nAChRs), and that of mRNAs encoding for alpha3, alpha4, alpha7 and beta2 nAChR subunits in both lines. The DA content of dialysates was measured before and after a 1-min perfusion of nicotine (6, 10 or 20 nmoles/min) and the resulting DA increase was taken as a measure of the alkaloid's intrinsic activity for nAChRs involved in the release of DA. The nicotine-induced increase of striatal DA release was greater in FSL than in FRL rats for all concentrations of nicotine, suggesting that the intrinsic activity of nicotine was greater in the FSL than in the FRL rats. This was further supported by our finding that the density of high-affinity nAChRs in the striatum of FSL rats was 44% greater than in the FRL rats, whereas affinity (K(D)) was virtually the same in the two lines of rats. Also the expression of mRNAs encoding for alpha(4), alpha(7), and beta(2) subunits in the striatum was greater in FSL than in FRL rats (attomol/microg total RNA, alpha(4):98+/-10 vs. 77+/-7; alpha(7):279+/-16 vs. 184+/-16; beta(2):310+/-19 vs. 201+/-12). We hypothesize that the difference in nicotine-induced DA release in the striatum of FSL and FRL rats depends on the difference in nAChR subunit expression in the striatum between the two lines. The Flinders rats could be used as a model for nicotine self-administration studies to evaluate the susceptibilities of FSL and FRL rats to nicotine dependence.

  20. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages

    SciTech Connect

    Kim, Mi Jin; Nepal, Saroj; Lee, Eung-Seok; Jeong, Tae Cheon; Kim, Sang-Hyun; Park, Pil-Hoon

    2013-11-15

    Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages. - Highlights: • Ethanol increases ROS production through up-regulation of Nox2 in macrophages. • Enhanced oxidative stress contributes to ethanol

  1. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines.

    PubMed

    Calmon, Marilia Freitas; Sichero, Laura; Boccardo, Enrique; Villa, Luisa Lina; Rahal, Paula

    2016-09-01

    Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16 E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. PMID:27240147

  2. Hydroxytyrosol Inhibits Cannabinoid CB1 Receptor Gene Expression in 3T3-L1 Preadipocyte Cell Line.

    PubMed

    Tutino, Valeria; Orlando, Antonella; Russo, Francesco; Notarnicola, Maria

    2016-02-01

    The 3T3-L1 preadipocyte cell line is a well characterized cell model for studying the adipocyte status and the molecular mechanisms involved in differentiation of these cells. 3T3-L1 preadipocytes have the ability to synthesize and degrade endocannabinoid anandamide (AEA) and their differentiation into adipocytes increases the expression of cannabinoid (CB1) and PPAR-γ receptors. Clinically, the blocking stimulation of the endocannabinoid pathway has been one of the first approaches proposed to counteract the obesity and obesity-associated diseases (such as diabetes, metabolic syndrome and cancer). In this connection, here we studied in cultured 3T3-L1 pre-adipocytes the effects of n-3-PUFA, α-Linolenic acid (OM-3), n-6-PUFA, Linoleic acid (OM-6), and hydroxytyrosol (HT) on the expression of CB1 receptor gene and the adipogenesis-related genes PPAR-γ, Fatty Acid Synthase (FAS) and Lipoprotein Lipase (LPL). HT was able to inhibit 3T3-L1 cell differentiation by down-regulating cell proliferation and CB1 receptor gene expression. HT exhibited anti-adipogenic effects, whereas OM-3 and OM-6 exerted an inhibitory action on cell proliferation associated with an induction of the preadipocytes differentiation and CB1 receptor gene expression. Moreover, the expression of FAS and LPL genes resulted increased after treatment with both HT and OM-3 and OM-6. The present study points out that the intake of molecules such as HT, contained in extra virgin olive oil, may be considered also in view of antiobesity and antineoplastic properties by acting directly on the adipose tissue and modulating CB1 receptor gene transcription.

  3. A combination of gene expression ranking and co-expression network analysis increases discovery rate in large-scale mutant screens for novel Arabidopsis thaliana abiotic stress genes.

    PubMed

    Ransbotyn, Vanessa; Yeger-Lotem, Esti; Basha, Omer; Acuna, Tania; Verduyn, Christoph; Gordon, Michal; Chalifa-Caspi, Vered; Hannah, Matthew A; Barak, Simon

    2015-05-01

    As challenges to food security increase, the demand for lead genes for improving crop production is growing. However, genetic screens of plant mutants typically yield very low frequencies of desired phenotypes. Here, we present a powerful computational approach for selecting candidate genes for screening insertion mutants. We combined ranking of Arabidopsis thaliana regulatory genes according to their expression in response to multiple abiotic stresses (Multiple Stress [MST] score), with stress-responsive RNA co-expression network analysis to select candidate multiple stress regulatory (MSTR) genes. Screening of 62 T-DNA insertion mutants defective in candidate MSTR genes, for abiotic stress germination phenotypes yielded a remarkable hit rate of up to 62%; this gene discovery rate is 48-fold greater than that of other large-scale insertional mutant screens. Moreover, the MST score of these genes could be used to prioritize them for screening. To evaluate the contribution of the co-expression analysis, we screened 64 additional mutant lines of MST-scored genes that did not appear in the RNA co-expression network. The screening of these MST-scored genes yielded a gene discovery rate of 36%, which is much higher than that of classic mutant screens but not as high as when picking candidate genes from the co-expression network. The MSTR co-expression network that we created, AraSTressRegNet is publicly available at http://netbio.bgu.ac.il/arnet. This systems biology-based screening approach combining gene ranking and network analysis could be generally applicable to enhancing identification of genes regulating additional processes in plants and other organisms provided that suitable transcriptome data are available. PMID:25370817

  4. A combination of gene expression ranking and co-expression network analysis increases discovery rate in large-scale mutant screens for novel Arabidopsis thaliana abiotic stress genes.

    PubMed

    Ransbotyn, Vanessa; Yeger-Lotem, Esti; Basha, Omer; Acuna, Tania; Verduyn, Christoph; Gordon, Michal; Chalifa-Caspi, Vered; Hannah, Matthew A; Barak, Simon

    2015-05-01

    As challenges to food security increase, the demand for lead genes for improving crop production is growing. However, genetic screens of plant mutants typically yield very low frequencies of desired phenotypes. Here, we present a powerful computational approach for selecting candidate genes for screening insertion mutants. We combined ranking of Arabidopsis thaliana regulatory genes according to their expression in response to multiple abiotic stresses (Multiple Stress [MST] score), with stress-responsive RNA co-expression network analysis to select candidate multiple stress regulatory (MSTR) genes. Screening of 62 T-DNA insertion mutants defective in candidate MSTR genes, for abiotic stress germination phenotypes yielded a remarkable hit rate of up to 62%; this gene discovery rate is 48-fold greater than that of other large-scale insertional mutant screens. Moreover, the MST score of these genes could be used to prioritize them for screening. To evaluate the contribution of the co-expression analysis, we screened 64 additional mutant lines of MST-scored genes that did not appear in the RNA co-expression network. The screening of these MST-scored genes yielded a gene discovery rate of 36%, which is much higher than that of classic mutant screens but not as high as when picking candidate genes from the co-expression network. The MSTR co-expression network that we created, AraSTressRegNet is publicly available at http://netbio.bgu.ac.il/arnet. This systems biology-based screening approach combining gene ranking and network analysis could be generally applicable to enhancing identification of genes regulating additional processes in plants and other organisms provided that suitable transcriptome data are available.

  5. Establishment of a canine mammary gland tumor cell line and characterization of its miRNA expression.

    PubMed

    Osaki, Tomohiro; Sunden, Yuji; Sugiyama, Akihiko; Azuma, Kazuo; Murahata, Yusuke; Tsuka, Takeshi; Ito, Norihiko; Imagawa, Tomohiro; Okamoto, Yoshiharu

    2016-09-30

    Canine mammary gland tumors (CMGTs), which are the most common neoplasms in sexually intact female dogs, have been suggested as a model for studying human breast cancer because of several similarities, including relative age of onset, risk factors, incidence, histological and molecular features, biological behavior, metastatic pattern, and responses to therapy. In the present study, we established a new cell line, the SNP cell line, from a CMGT. A tumor formed in each NOD.CB17-Prkdc(scid)/J mouse at the site of subcutaneous SNP cell injection. SNP cells are characterized by proliferation in a tubulopapillary pattern and are vimentin positive. Moreover, we examined miRNA expression in the cultured cells and found that the expression values of miRNA-143 and miRNA-138a showed the greatest increase and decrease, respectively, of all miRNAs observed, indicating that these miRNAs might play a significant role in the malignancy of SNP cells. Overall, the results of this study indicate that SNP cells might serve as a model for future genetic analysis and clinical treatments of human breast tumors.

  6. Effect of bixin and norbixin on the expression of cytochrome P450 in HepG2 cell line.

    PubMed

    Matuo, Míriam Cristina Sakuragui; de Oliveira Takamoto, Rafael Teruiti; Kikuchi, Irene Satiko; de Jesus Andreoli Pinto, Terezinha

    2013-08-01

    Bixin and norbixin are the main components of annatto, which is extracted from Bixa orellana and largely used as natural colorant in the food and pharmaceutical industries. Annatto can enhance CYP1A and CYP2B activity in rats; however, the inducer effect has not been investigated in human cell lines. In this study, the ability of bixin and norbixin to induce the cytochrome P450 (CYP) enzymes was assessed in HepG2 human hepatoma cell line. HepG2 cells were treated with bixin and norbixin, and the expression of the CYP genes quantified by real-time reverse transcription polymerase chain reaction (RT-PCR). Expression of CYP1A1 and CYP1A2 was significantly increased by bixin treatment, while CYP2B6, 2C9, 2E1 and 3A4 were unaffected. Cells were treated with norbixin showed no inducer effect. The results suggest that the inducer potential of annatto is attributed to bixin, but not to norbixin, despite their similarities in molecular structure. PMID:23554079

  7. Role of plasma membrane lipid composition on cellular homeostasis: learning from cell line models expressing fatty acid desaturases

    PubMed Central

    Jaureguiberry, María S.; Tricerri, M. Alejandra; Sanchez, Susana A.; Finarelli, Gabriela S.; Montanaro, Mauro A.; Prieto, Eduardo D.; Rimoldi, Omar J.

    2014-01-01

    Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by regulating PM organization and Chol exportation and homeostasis. PMID:24473084

  8. Establishment of a canine mammary gland tumor cell line and characterization of its miRNA expression

    PubMed Central

    Sunden, Yuji; Sugiyama, Akihiko; Azuma, Kazuo; Murahata, Yusuke; Tsuka, Takeshi; Ito, Norihiko; Imagawa, Tomohiro; Okamoto, Yoshiharu

    2016-01-01

    Canine mammary gland tumors (CMGTs), which are the most common neoplasms in sexually intact female dogs, have been suggested as a model for studying human breast cancer because of several similarities, including relative age of onset, risk factors, incidence, histological and molecular features, biological behavior, metastatic pattern, and responses to therapy. In the present study, we established a new cell line, the SNP cell line, from a CMGT. A tumor formed in each NOD.CB17-Prkdcscid/J mouse at the site of subcutaneous SNP cell injection. SNP cells are characterized by proliferation in a tubulopapillary pattern and are vimentin positive. Moreover, we examined miRNA expression in the cultured cells and found that the expression values of miRNA-143 and miRNA-138a showed the greatest increase and decrease, respectively, of all miRNAs observed, indicating that these miRNAs might play a significant role in the malignancy of SNP cells. Overall, the results of this study indicate that SNP cells might serve as a model for future genetic analysis and clinical treatments of human breast tumors. PMID:26726024

  9. Mycoplasma arthritidis-derived superantigen induces proinflammatory monokine gene expression in the THP-1 human monocytic cell line.

    PubMed Central

    al-Daccak, R; Mehindate, K; Hébert, J; Rink, L; Mecheri, S; Mourad, W

    1994-01-01

    Soluble factors produced by Mycoplasma arthritidis play an important role in the pathology of arthritis in rodents, which closely resembles human rheumatoid arthritis. At least one of the products of these microorganisms, M. arthritidis-T cell mitogen (MAM), has biological activities in common with superantigens. These superantigens activate T cells in a V beta-restricted fashion, and this response is strictly dependent on the presence of major histocompatibility complex (MHC) class II-positive cells. In the present study, we have examined the ability of MAM to induce proinflammatory monokine (interleukin 1 beta [IL-1 beta] and tumor necrosis factor alpha [TNF-alpha]) gene expression in the THP-1 monocytic cell line. Treatment of these cells (which express a very low level of HLA-DR molecules) with gamma interferon (INF-gamma) induced HLA-DR, -DQ, and -DP molecules and enabled them to respond to MAM in a dose-dependent manner, resulting in an increase in the level of steady-state mRNA for IL-1 beta and TNF-alpha. Stimulation of the U937 monocytic cell line (MHC class II-negative even after INF-gamma treatment) with MAM did not induce either IL-1 beta or TNF-alpha transcription. Moreover, MAM adsorption on Raji (MHC class II-positive) cells resulted in the loss of its cytokine-inducing activity to induce monokine gene expression. These findings demonstrate clearly that MAM induces monokine gene expression following interaction with MHC class II molecules. Pretreatment of INF-gamma-treated THP-1 cells with the transcription inhibitor actinomycin D prevented the induction of monokine mRNA, whereas cycloheximide superinduced mRNA after stimulation with MAM. Finally, our results, obtained with protein tyrosine kinase inhibitors and antiphosphotyrosine Western blotting (immunoblotting), indicate that protein tyrosine kinase is involved in MAM-induced IL-1 beta and TNF-alpha gene expression in the THP-1 monocytic cell line. The capacity of MAM to induce proinflammatory

  10. Decrease in drug accumulation and in tumour aggressiveness marker expression in a fenretinide-induced resistant ovarianumour cell line

    PubMed Central

    Appierto, V; Cavadini, E; Pergolizzi, R; Cleris, L; Lotan, R; Canevari, S; Formelli, F

    2001-01-01

    We investigated whether the efficacy of fenretinide (HPR) against ovarian tumours may be limited by induction of resistance. The human ovarian carcinoma cell line A2780, which is sensitive to a pharmacologically achievable HPR concentration (IC 50= 1 μM), became 10-fold more resistant after exposure to increasing HPR concentrations. The cells (A2780/HPR) did not show cross-resistance to the synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and were not sensitive, similarly to the parent line, to all- trans -retinoic acid, 13- cis -retinoic acid or N-(4-methoxyphenyl)retinamide. A2780/HPR cells showed, compared to parental cells, a 3-fold reduction in colony-forming ability in agar. The development of HPR resistance was associated with a marked increase in retinoic acid receptor β (RARβ) mRNA and protein levels, which decreased, together with drug resistance, after drug removal. The expression of cell surface molecules associated with tumour progression including HER-2, laminin receptor and β1 integrin was markedly reduced. The increase in the levels of reactive oxygen species is not involved in HPR-resistance because it was similar in parental and resistant cells. Conversely differences in pharmacokinetics may account for resistance because, in A2780/HPR cells, intracellular peak drug levels were 2 times lower than in A2780 cells and an as yet unidentified polar metabolite was present. These data suggest that acquired resistance to HPR is associated with changes in marker expression, suggestive of a more differentiated status and may be explained, at least in part, by reduced drug accumulation and increased metabolism. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11384104

  11. Increased Expression of miR-23a Mediates a Loss of Expression in the RAF Kinase Inhibitor Protein RKIP

    PubMed Central

    Hatzl, Stefan; Geiger, Olivia; Kuepper, Maja Kim; Caraffini, Veronica; Seime, Till; Furlan, Tobias; Nussbaumer, Erika; Wieser, Rotraud; Pichler, Martin; Scheideler, Marcel; Nowek, Katarzyna; Jongen-Lavrencic, Mojca; Quehenberger, Franz; Wölfler, Albert; Troppmair, Jakob; Sill, Heinz; Zebisch, Armin

    2016-01-01

    RAF kinase inhibitor protein (RKIP) is a seminal regulator of intracellular signaling and exhibits both antimetastatic and antitumorigenic properties. Decreased expression of RKIP has been described in several human malignancies, including acute myelogenous leukemia (AML). As the mechanisms leading to RKIP loss in AML are still unclear, we aimed to analyze the potential involvement of miRNAs within this study. miRNA microarray and qPCR data of more than 400 AML patient specimens revealed correlation between decreased expression of RKIP and increased expression of miR-23a, a member of the miR-23a/27a/24-2 cluster. In functional experiments, overexpression of miR-23a decreased RKIP mRNA and protein expression, whereas miR-23a inhibition caused the opposite effect. By using an RKIP 3′-untranslated region luciferase reporter construct with and without mutation or deletion of the putative miR-23a–binding site, we could show that RKIP modulation by miR-23a is mediated via direct binding to this region. Importantly, miR-23a overexpression induced a significant increase of proliferation in hematopoietic cells. Simultaneous transfection of an RKIP expression construct lacking the miR-23a–binding sites reversed this phenotype, indicating that this effect is truly mediated via downregulation of RKIP. Finally, by analyzing more than 4,300 primary patient specimens via database retrieval from The Cancer Genome Atlas, we could highlight the importance of the miR-23a/RKIP axis in a broad range of human cancer entities. In conclusion, we have identified miR-23a as a negative regulator of RKIP expression in AML and have provided data that suggest the importance of our observation beyond this tumor entity. PMID:27197200

  12. Aerosol-induced brucellosis increases TLR-2 expression and increased complexity in the microanatomy of astroglia in rhesus macaques.

    PubMed

    Lee, Kim M; Chiu, Kevin B; Sansing, Hope A; Didier, Peter J; Ficht, Thomas A; Arenas-Gamboa, Angela M; Roy, Chad J; Maclean, Andrew G

    2013-01-01

    Brucella melitensis, a bacterial pathogen and agent of epizootic abortion causes multiple pathologies in humans as well as a number of agriculturally important animal species. Clinical human brucellosis manifests as a non-specific, chronic debilitating disease characterized by undulant fever, arthropathies, cardiomyopathies and neurological sequelae. These symptoms can occur acutely for a few weeks or persist for months to years. Within the brain, endothelial and glial cells can be infected leading to downstream activation events including matrix metalloprotease (MMP) and cytokine secretion and Toll-like receptor (TLR) signaling. These events are likely to lead to tissue remodeling, including morphologic changes in neuronal and glial cells, which are linked to neurological complications including depressive behavior, immune activation and memory loss. Our hypothesis was that B. melitensis infection and neurobrucellosis would lead to activation of astrocytes through upregulation of TLR2 and stimulate concurrent changes in the microanatomy. All six animals were infected via inhalation route. TLR2 expression was approximately doubled in white matter astrocytes of infected rhesus macaques. There was also a 50% increase in the number of astrocytes per unit area in subcortical white matter tracts suggesting increased innate immune activation. This coincided with dramatic increases in the length and complexity of the cell arbor of hypertrophic astrocytes in both cortical gray and white matter. Thus, aerosol-induced brucellosis results in dramatically increased innate immune activation of astrocytes in the absence of widespread neuroinflammation. PMID:24350061

  13. Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression

    PubMed Central

    Henry, Matthew K.; Welliver, Kathryn C.; Jepson, Amanda J.; Garnett, Emily R.

    2013-01-01

    Hindlimb unloading (HU) is a well-established animal model of cardiovascular deconditioning. Previous data indicate that HU results in cardiac sympathovagal imbalance. It is well established that cardiac sympathovagal imbalance increases the risk for developing cardiac arrhythmias. The cardiac gap junction protein connexin 43 (Cx43) is predominately expressed in the left ventricle (LV) and ensures efficient cell-to-cell electrical coupling. In the current study we wanted to test the hypothesis that HU would result in increased predisposition to cardiac arrhythmias and alter the expression and/or phosphorylation of LV-Cx43. Electrocardiographic data using implantable telemetry were obtained over a 10- to 14-day HU or casted control (CC) condition and in response to a sympathetic stressor using isoproterenol administration and brief restraint. The arrhythmic burden was calculated using a modified scoring system to quantify spontaneous and provoked arrhythmias. In addition, Western blot analysis was used to measure LV-Cx43 expression in lysates probed with antibodies directed against the total and an unphosphorylated form of Cx43 in CC and HU rats. HU resulted in a significantly greater total arrhythmic burden during the sympathetic stressor with significantly more ventricular arrhythmias occurring. In addition, there was increased expression of total LV-Cx43 observed with no difference in the expression of unphosphorylated LV-Cx43. Specifically, the increased expression of LV-Cx43 was consistent with the phosphorylated form. These data taken together indicate that cardiovascular deconditioning produced through HU results in increased predisposition to cardiac arrhythmias and increased expression of phosphorylated LV-Cx43. PMID:23302960

  14. Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression.

    PubMed

    Moffitt, Julia A; Henry, Matthew K; Welliver, Kathryn C; Jepson, Amanda J; Garnett, Emily R

    2013-03-01

    Hindlimb unloading (HU) is a well-established animal model of cardiovascular deconditioning. Previous data indicate that HU results in cardiac sympathovagal imbalance. It is well established that cardiac sympathovagal imbalance increases the risk for developing cardiac arrhythmias. The cardiac gap junction protein connexin 43 (Cx43) is predominately expressed in the left ventricle (LV) and ensures efficient cell-to-cell electrical coupling. In the current study we wanted to test the hypothesis that HU would result in increased predisposition to cardiac arrhythmias and alter the expression and/or phosphorylation of LV-Cx43. Electrocardiographic data using implantable telemetry were obtained over a 10- to 14-day HU or casted control (CC) condition and in response to a sympathetic stressor using isoproterenol administration and brief restraint. The arrhythmic burden was calculated using a modified scoring system to quantify spontaneous and provoked arrhythmias. In addition, Western blot analysis was used to measure LV-Cx43 expression in lysates probed with antibodies directed against the total and an unphosphorylated form of Cx43 in CC and HU rats. HU resulted in a significantly greater total arrhythmic burden during the sympathetic stressor with significantly more ventricular arrhythmias occurring. In addition, there was increased expression of total LV-Cx43 observed with no difference in the expression of unphosphorylated LV-Cx43. Specifically, the increased expression of LV-Cx43 was consistent with the phosphorylated form. These data taken together indicate that cardiovascular deconditioning produced through HU results in increased predisposition to cardiac arrhythmias and increased expression of phosphorylated LV-Cx43.

  15. Tianeptine increases brain-derived neurotrophic factor expression in the rat amygdala.

    PubMed

    Reagan, Lawrence P; Hendry, Robert M; Reznikov, Leah R; Piroli, Gerardo G; Wood, Gwendolyn E; McEwen, Bruce S; Grillo, Claudia A

    2007-06-22

    Chronic restraint stress affects hippocampal and amygdalar synaptic plasticity as determined by electrophysiological, morphological and behavioral measures, changes that are inhibited by some but not all antidepressants. The efficacy of some classes of antidepressants is proposed to involve increased phosphorylation of cAMP response element binding protein (CREB), leading to increased expression of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF). Conversely, some studies suggest that acute and chronic stress downregulate BDNF expression and activity. Accordingly, the aim of the current study was to examine total and phosphorylated CREB (pCREB), as well as BDNF mRNA and protein levels in the hippocampus and amygdala of rats subjected to chronic restraint stress in the presence and absence of the antidepressant tianeptine. In the hippocampus, chronic restraint stress increased pCREB levels without affecting BDNF mRNA or protein expression. Tianeptine administration had no effect upon these measures in the hippocampus. In the amygdala, BDNF mRNA expression was not modulated in chronic restraint stress rats given saline in spite of increased pCREB levels. Conversely, BDNF mRNA levels were increased in the amygdala of chronic restraint stress/tianeptine rats in the absence of changes in pCREB levels when compared to non-stressed controls. Amygdalar BDNF protein increased while pCREB levels decreased in tianeptine-treated rats irrespective of stress conditions. Collectively, these results demonstrate that tianeptine concomitantly decreases pCREB while increasing BDNF expression in the rat amygdala, increases in neurotrophic factor expression that may participate in the enhancement of amygdalar synaptic plasticity mediated by tianeptine.

  16. Increased longevity mediated by yeast NDI1 expression in Drosophila intestinal stem and progenitor cells

    PubMed Central

    Hur, Jae H.; Bahadorani, Sepehr; Graniel, Jacqueline; Koehler, Christopher L.; Ulgherait, Matthew; Rera, Michael; Jones, D. Leanne; Walker, David W.

    2013-01-01

    A functional decline in tissue stem cells and mitochondrial dysfunction have each been linked to aging and multiple aging-associated pathologies. However, the interplay between energy homeostasis, stem cells, and organismal aging remains poorly understood. Here, we report that expression of the single-subunit yeast alternative NADH dehydrogenase, ndi1, in Drosophila intestinal stem and progenitor cells delays the onset of multiple markers of intestinal aging and extends lifespan. In addition, expression of ndi1 in the intestine increases feeding behavior and results in organismal weight gain. Consistent with increased nutrient uptake, flies expressing ndi1 in the digestive tract display a systemic reduction in the activity of AMP-activated protein kinase (AMPK), a key cellular energy sensor. Together, these results demonstrate that ndi1 expression in the intestinal epithelium is an effective strategy to delay tissue and organismal aging. PMID:24038661

  17. Transgene expression and silencing in a tick cell line: A model system for functional tick genomics.

    PubMed

    Kurtti, Timothy J; Mattila, Joshua T; Herron, Michael J; Felsheim, Roderick F; Baldridge, Gerald D; Burkhardt, Nicole Y; Blazar, Bruce R; Hackett, Perry B; Meyer, Jason M; Munderloh, Ulrike G

    2008-10-01

    The genome project of the black legged tick, Ixodes scapularis, provides sequence data for testing gene function and regulation in this important pathogen vector. We tested Sleeping Beauty (SB), a Tc1/mariner group transposable element, and cationic lipid-based transfection reagents for delivery and genomic integration of transgenes into I. scapularis cell line ISE6. Plasmid DNA and dsRNA were effectively transfected into ISE6 cells and they were successfully transformed to express a red fluorescent protein (DsRed2) and a selectable marker, neomycin phosphotransferase (NEO). Frequency of transformation was estimated as 1 transformant per 5000-10,000 cells and cultures were incubated for 2-3 months in medium containing the neomycin analog G418 in order to isolate transformants. Genomic integration of the DsRed2 transgene was confirmed by inverse PCR and sequencing that demonstrated a TA nucleotide pair inserted between SB inverted/direct repeat sequences and tick genomic sequences, indicating that insertion of the DsRed2 gene into the tick cell genome occurred through the activity of SB transposase. RNAi using dsRNA transcribed from the DsRed2 gene silenced expression of red fluorescent protein in transformed ISE6 cells. SB transposition in cell line ISE6 provides an effective means to explore the functional genomics of I. scapularis. PMID:18722527

  18. The Ah receptor regulates growth factor expression in head and neck squamous cell carcinoma cell lines.

    PubMed

    John, Kaarthik; Lahoti, Tejas S; Wagner, Kelly; Hughes, Jarod M; Perdew, Gary H

    2014-10-01

    Previous studies in head and neck squamous cell carcinoma (HNSCC) cell lines have revealed that the Ah receptor (AHR) plays a significant role in mediating the "aggressive" phenotype of these cells, which includes enhanced inflammatory signaling (e.g., IL6) and migratory potential. Here we sought to identify putative novel targets of the AHR associated with enhanced tumor invasiveness. Global gene expression analysis identified a number of genes that are repressed upon treatment of OSC-19 or HN30 cells with an AHR antagonist. Three growth factors were targets of AHR activity; amphiregulin (AREG), epiregulin (EREG), and platelet-derived growth factor A (PDGFA) were repressed by an AHR antagonist and further examined. Quantitative PCR analysis, ELISA, and siRNA-mediated knock down of AHR revealed an attenuation of basal and/or induced levels of expression of these growth factors in two HNSCC lines, following AHR antagonism. In silico analysis revealed that these growth factors possess dioxin-like response elements. Two other AHR ligands, 6-formylindolo[3,2-b]carbazole and benzo(a)pyrene (BP) also elicited similar responses. In conclusion, this study identified AREG, EREG, and PDGFA as growth factor targets of AHR activity associated with metastatic phenotype of HNSCC cells, suggesting that attenuation of AHR activity may be a therapeutic strategy.

  19. Increased expression of argininosuccinate synthetase protein predicts poor prognosis in human gastric cancer.

    PubMed

    Shan, Yan-Shen; Hsu, Hui-Ping; Lai, Ming-Derg; Yen, Meng-Chi; Luo, Yi-Pey; Chen, Yi-Ling

    2015-01-01

    Aberrant expression of argininosuccinate synthetase (ASS1, also known as ASS) has been found in cancer cells and is involved in the carcinogenesis of gastric cancer. The aim of the present study was to investigate the level of ASS expression in human gastric cancer and to determine the possible correlations between ASS expression and clinicopathological findings. Immunohistochemistry was performed on paraffin‑embedded tissues to determine whether ASS was expressed in 11 of 11 specimens from patients with gastric cancer. The protein was localized primarily to the cytoplasm of cancer cells and normal epithelium. In the Oncomine cancer microarray database, expression of the ASS gene was significantly increased in gastric cancer tissues. To investigate the clinicopathological and prognostic roles of ASS expression, we performed western blot analysis of 35 matched specimens of gastric adenocarcinomas and normal tissue obtained from patients treated at the National Cheng Kung University Hospital. The ratio of relative ASS expression (expressed as the ASS/β-actin ratio) in tumor tissues to that in normal tissues was correlated with large tumor size (P=0.007) and with the tumor, node, metastasis (TNM) stage of the American Joint Committee on Cancer staging system (P=0.031). Patients whose cancer had increased the relative expression of ASS were positive for perineural invasion and had poor recurrence-free survival. In summary, ASS expression in gastric cancer was associated with a poor prognosis. Further study of mechanisms to silence the ASS gene or decrease the enzymatic activity of ASS protein has the potential to provide new treatments for patients with gastric cancer.

  20. Evaluation of cytokine gene expression after avian influenza virus infection in avian cell lines and primary cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The innate immune responses elicited by avian influenza virus (AIV) infection has been studied by measuring cytokine gene expression by relative real time PCR (rRT-PCR) in vitro, using both cell lines and primary cell cultures. Continuous cell lines offer advantages over the use of primary cell cult...

  1. Increased aquaporin 1 and 5 membrane expression in the lens epithelium of cataract patients.

    PubMed

    Barandika, Olatz; Ezquerra-Inchausti, Maitane; Anasagasti, Ander; Vallejo-Illarramendi, Ainara; Llarena, Irantzu; Bascaran, Lucia; Alberdi, Txomin; De Benedetti, Giacomo; Mendicute, Javier; Ruiz-Ederra, Javier

    2016-10-01

    In this work we have analyzed the expression levels of the main aquaporins (AQPs) expressed in human lens epithelial cells (HLECs) using 112 samples from patients treated with cataract surgery and 36 samples from individuals treated with refractive surgery, with transparent lenses as controls. Aquaporin-1 (AQP1) is the main AQP, representing 64.1% of total AQPs in HLECs, with aquaporin-5 (AQP5) representing 35.9% in controls. A similar proportion of each AQP in cataract was found. Although no differences were found at the mRNA level compared to controls, a significant 1.65-fold increase (p=0.001) in AQP1protein expression was observed in HLECs from cataract patients, with the highest differences being found for nuclear cataracts (2.1-fold increase; p<0.001). A similar trend was found for AQP5 (1.47-fold increase), although the difference was not significant (p=0.161). Moreover we have shown increased membrane AQP5 protein expression in HLECs of patients with cataracts. No association of AQP1 or AQP5 expression levels with age or sex was observed in either group. Our results suggest regulation of AQP1 and AQP5 at the post-translational level and support previous observations on the implication of AQP1 and 5 in maintenance of lens transparency in animal models. Our results likely reflect a compensatory response of the crystalline lens to delay cataract formation by increasing the water removal rate.

  2. Increased aquaporin 1 and 5 membrane expression in the lens epithelium of cataract patients.

    PubMed

    Barandika, Olatz; Ezquerra-Inchausti, Maitane; Anasagasti, Ander; Vallejo-Illarramendi, Ainara; Llarena, Irantzu; Bascaran, Lucia; Alberdi, Txomin; De Benedetti, Giacomo; Mendicute, Javier; Ruiz-Ederra, Javier

    2016-10-01

    In this work we have analyzed the expression levels of the main aquaporins (AQPs) expressed in human lens epithelial cells (HLECs) using 112 samples from patients treated with cataract surgery and 36 samples from individuals treated with refractive surgery, with transparent lenses as controls. Aquaporin-1 (AQP1) is the main AQP, representing 64.1% of total AQPs in HLECs, with aquaporin-5 (AQP5) representing 35.9% in controls. A similar proportion of each AQP in cataract was found. Although no differences were found at the mRNA level compared to controls, a significant 1.65-fold increase (p=0.001) in AQP1protein expression was observed in HLECs from cataract patients, with the highest differences being found for nuclear cataracts (2.1-fold increase; p<0.001). A similar trend was found for AQP5 (1.47-fold increase), although the difference was not significant (p=0.161). Moreover we have shown increased membrane AQP5 protein expression in HLECs of patients with cataracts. No association of AQP1 or AQP5 expression levels with age or sex was observed in either group. Our results suggest regulation of AQP1 and AQP5 at the post-translational level and support previous observations on the implication of AQP1 and 5 in maintenance of lens transparency in animal models. Our results likely reflect a compensatory response of the crystalline lens to delay cataract formation by increasing the water removal rate. PMID:27497833

  3. The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule

    PubMed Central

    1982-01-01

    Monoclonal antibodies were prepared against the Madin-Darby canine kidney (MDCK) cell line to identify epithelial cell surface macromolecules involved in renal function. Lymphocyte hybrids were generated by fusing P3U-1 myeloma cells with spleen cells from a C3H mouse immunized with MDCK cells. Hybridomas secreting anti-MDCK antibodies were obtained and clonal lines isolated in soft agarose. We are reporting on one hybridoma line that secretes a monoclonal antibody that binds to MDCK cells at levels 20-fold greater than background binding. Indirect immunofluorescence microscopy was utilized to study the distribution of antibody binding on MDCK cells and on frozen sections of dog kidney and several nonrenal tissues. In the kidney the fluorescence staining pattern demonstrates that the antibody recognizes an antigenic determinant that is expressed only on the epithelial cells of the thick ascending limb of Henle's loops and the distal convoluted tubule and appears to be localized on the basolateral plasma membrane. This antigen also has a unique distribution in non-renal tissues and can only be detected on cells known to be active in transepithelial ion movements. These results indicate the probable distal tubule origin of MDCK and suggest that the monoclonal antibody recognizes a cell surface antigen involved in physiological functions unique to the kidney distal tubule and transporting epithelia of nonrenal tissues. PMID:6178742

  4. Increased fibronectin expression in sturge-weber syndrome fibroblasts and brain tissue.

    PubMed

    Comi, Anne M; Hunt, Piper; Vawter, Marquis P; Pardo, Carlos A; Becker, Kevin G; Pevsner, Jonathan

    2003-05-01

    Sturge-Weber syndrome (SWS) is a neurocutaneous disorder that presents with a facial port-wine stain and a leptomeningeal angioma. Fibronectin expression regulates angiogenesis and vasculogenesis and participates in brain tissue responses to ischemia and seizures. We therefore hypothesized that abnormal gene expression of fibronectin and other extracellular matrix genes would be found in SWS brain tissue and SWS port-wine skin fibroblasts. Fibronectin gene and protein expression from port-wine-derived fibroblasts were compared with that from normal skin-derived fibroblasts of four individuals with SWS using microarrays, reverse transcriptase-PCR, Western analysis, and immunocytochemistry. Fibronectin gene and/or protein expression from eight SWS surgical brain samples was compared with that in two surgical epilepsy brain samples and six postmortem brain samples using microarrays, reverse transcriptase-PCR, and Western analysis. The gene expression of fibronectin was significantly increased (p < 0.05) in the SWS port-wine-derived fibroblasts compared with that of fibroblasts from SWS normal skin. A trend for increased protein levels of fibronectin in port-wine fibroblasts was found by Western analysis. No difference in the pattern of fibronectin staining was detected. The gene expression of fibronectin was significantly increased (p < 0.05), and a trend for increased fibronectin protein expression was found in the SWS surgical brain samples compared with the postmortem controls. These results suggest a potential role for fibronectin in the pathogenesis of SWS and in the brain's response to chronic ischemic injury in SWS. The reproducible differences in fibronectin gene expression between the SWS port-wine-derived fibroblasts and the SWS normal skin-derived fibroblasts are consistent with the presence of a hypothesized somatic mutation underlying SWS. PMID:12621118

  5. Ultraviolet B irradiation increases endothelin-1 and endothelin receptor expression in cultured human keratinocytes.

    PubMed

    Tsuboi, R; Sato, C; Oshita, Y; Hama, H; Sakurai, T; Goto, K; Ogawa, H

    1995-09-01

    The effect of ultraviolet B (UVB) irradiation on endothelin-1 (ET-1) and ET receptor expression was examined using cultured normal human keratinocytes. Keratinocytes secreted ET-1 in the medium at a level of 2.1 pg/day/10(5) cells. UVB irradiation up to 10 mJ/cm2 increased ET-1 secretion 3-fold, and potentiated expression of mRNA for ET-1. Both ETA and ETB receptor mRNAs were detected in keratinocytes, and their expression was up-regulated by 5 mJ/cm2 UVB irradiation.

  6. High glucose and palmitate increases bone morphogenic protein 4 expression in human endothelial cells

    PubMed Central

    Hong, Oak-Kee; Yoo, Soon-Jib; Son, Jang-Won; Kim, Mee-Kyoung; Baek, Ki-Hyun; Song, Ki-Ho; Cha, Bong-Yun; Jo, Hanjoong

    2016-01-01

    Here, we investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) aff ect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this eff ect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a proinflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of infl ammatory adhesion molecules and ROS production. Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL. PMID:26937213

  7. High glucose and palmitate increases bone morphogenic protein 4 expression in human endothelial cells.

    PubMed

    Hong, Oak-Kee; Yoo, Soon-Jib; Son, Jang-Won; Kim, Mee-Kyoung; Baek, Ki-Hyun; Song, Ki-Ho; Cha, Bong-Yun; Jo, Hanjoong; Kwon, Hyuk-Sang

    2016-03-01

    Here, we investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) aff ect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this eff ect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a proinflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of infl ammatory adhesion molecules and ROS production. Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL. PMID:26937213

  8. Divergent control of Cav-1 expression in non-cancerous Li-Fraumeni syndrome and human cancer cell lines

    PubMed Central

    Sherif, Zaki A.; Sultan, Ahmed S.

    2013-01-01

    Li-Fraumeni syndrome (LFS) is primarily characterized by development of tumors exhibiting germ-line mutations in the p53 gene. Cell lines developed from patients of a LFS family have decreased p53 activity as evidenced by the absence of apoptosis upon etoposide treatment. To test our hypothesis that changes in gene expression beyond p53 per se are contributing to the development of tumors, we compared gene expression in non-cancerous skin fibroblasts of LFS-affected (p53 heterozygous) vs. non-affected (p53 wild-type homozygous) family members. Expression analysis showed that several genes were differentially regulated in the p53 homozygous and heterozygous cell lines. We were particularly intrigued by the decreased expression (~88%) of a putative tumor-suppressor protein, caveolin-1 (Cav-1), in the p53-mutant cells. Decreased expression of Cav-1 was also seen in both p53-knockout and p21-knockout HTC116 cells suggesting that p53 controls Cav-1 expression through p21 and leading to the speculation that p53, Cav-1 and p21 may be part of a positive auto-regulatory feedback loop. The direct relationship between p53 and Cav-1 was also tested with HeLa cells (containing inactive p53), which expressed a significantly lower Cav-1 protein. A panel of nonfunctional and p53-deficient colon and epithelial breast cancer cell lines showed undetectable expression of Cav-1 supporting the role of p53 in the control of Cav-1. However, in two aggressively metastasizing breast cancer cell lines, Cav-1 was strongly expressed suggesting a possible role in tumor metastasis. Thus, there is a divergent control of Cav-1 expression as evidenced in non-cancerous Li-Fraumeni syndrome and some aggressive human cancer cell lines. PMID:23114650

  9. Characterization of transgenic zebrafish lines that express GFP in the retina, pineal gland, olfactory bulb, hatching gland, and optic tectum.

    PubMed

    Fang, Wei; Bonaffini, Sarah; Zou, Jian; Wang, Xiaolei; Zhang, Cen; Tsujimura, Taro; Kawamura, Shoji; Wei, Xiangyun

    2013-01-01

    Transgenic animals are powerful tools to study gene function invivo. Here we characterize several transgenic zebrafish lines that express green fluorescent protein (GFP) under the control of the LCR(RH2)-RH2-1 or LCR(RH2)-RH2-2 green opsin regulatory elements. Using confocal immunomicroscopy, stereo-fluorescence microscopy, and Western blotting, we show that the Tg(LCR(RH2)-RH2-1:GFP)(pt112) and Tg(LCR(RH2)-RH2-2:GFP)(pt115) transgenic zebrafish lines express GFP in the pineal gland and certain types of photoreceptors. In addition, some of these lines also express GFP in the hatching gland, optic tectum, or olfactory bulb. Some of the expression patterns differ significantly from previously published similar transgenic fish lines, making them useful tools for studying the development of the corresponding tissues and organs. In addition, the variations of GFP expression among different lines corroborate the notion that transgenic expression is often subjected to position effect, thus emphasizing the need for careful verification of expression patterns when transgenic animal models are utilized for research.

  10. Investigation of Astragalus honey and propolis extract's cytotoxic effect on two human cancer cell lines and their oncogen and proapoptotic gene expression profiles

    PubMed Central

    Sadeghi-Aliabadi, Hojjat; Hamzeh, Jamal; Mirian, Mina

    2015-01-01

    Background: Cancer is one of the major fatal human diseases. Natural products have been used in the treatment of cancer for long time. Bee products including honey and propolis have been introduced for malignancy treatment in recent decades. In this study cytotoxicity of bee products and their effects on the expression of proapoptotic genes have been investigated. Materials and Methods: Cytotoxic effects of Astragalus honey, ethanol extract of propolis and a sugar solution (as control) against HepG2, 5637 and L929 cell lines have been evaluated by the MTT assay. Total RNAs of treated cells were isolated and p53 and Bcl-2 gene expression were evaluated, using real-time PCR. Results: Propolis IC50 values were 58, 30 and 15 μg/ml against L929, HepG2 and 5637, respectively. These values for honey were 3.1%, 2.4% and 1.9%, respectively. Propolis extract has increased the expression of the Bcl-2 gene in all cell lines whereas the honey decreased that significantly (P < 0.05). Also, we found that honey and propolis decreased p53 gene expression in HepG2 and 5637 significantly but not in L929 cells. The sugar solution increased the expression of p53 in two cancer cell lines but no significant changes were observed in the expression of this gene in L929 as normal mouse cell. Conclusion: By downregulation of Bcl-2 expression it could be concluded that the cytotoxicity of honey was more than two fold against tested cancer cells compared with the sugar solution. No significant changes were observed in the expression of p53 in honey-treated cells. Propolis had no significant effect on Bcl-2 and p53 gene expressions (P > 0.05). PMID:25789268

  11. In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor.

    PubMed Central

    Damstrup, L.; Rude Voldborg, B.; Spang-Thomsen, M.; Brünner, N.; Skovgaard Poulsen, H.

    1998-01-01

    Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16% of the cells added to the upper chamber were able to traverse the Matrigel membrane. Expression of several matrix metalloproteases (MMP), of tissue inhibitor of MMP (TIMP) and of cathepsin B was evaluated by immunoprecipitation, Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential of SCLC cell lines. Images Figure 1 Figure 3 Figure 4 PMID:9744504

  12. Common Spatial Organization of Number and Emotional Expression: A Mental Magnitude Line

    ERIC Educational Resources Information Center

    Holmes, Kevin J.; Lourenco, Stella F.

    2011-01-01

    Converging behavioral and neural evidence suggests that numerical representations are mentally organized in left-to-right orientation. Here we show that this format of spatial organization extends to emotional expression. In Experiment 1, right-side responses became increasingly faster as number (represented by Arabic numerals) or happiness…

  13. MMP-9 expression increases according to the grade of squamous intraepithelial lesion in cervical smears.

    PubMed

    Matheus, Erika R; Zonta, Marco A; Discacciati, Michelle G; Paruci, Priscila; Velame, Fernanda; Cardeal, Laura B S; Barros, Silvia B M; Pignatari, Antonio C; Maria-Engler, Silvya S

    2014-10-01

    Studies about cervical carcinogenesis have demonstrated the increased expression of matrix-metalloproteinase (MMP) according to the grade of cervical intraepithelial lesions. Considering the importance of innovative techniques to introduce noninvasive and rapid diagnoses for patients, this study aimed to perform MMP-9 immunocytochemistry in cervical smears according to the cytopathological diagnoses, in order to monitor MMP activity in cervical smears. This cross-sectional study investigated the expression of MMP-9 in normal cervical smears, inflammatory cervical smears, squamous intraepithelial lesions, and cervical carcinoma. Cervical smears from 630 women were collected for cytopathological diagnoses and immunocytochemistry. Women with squamous intraepithelial lesions showed an increase in MMP-9 expression, with moderate to intense staining occurring with increasing cervical lesion grade. The prevalence of moderate to intense MMP-9 staining was 9% in normal cervical smears, 12% in cervical inflammation, 24% in low-grade squamous intraepithelial lesion (LSIL), 92% in high-grade squamous intraepithelial lesions (HSIL) and 100% in cervical carcinoma cases. In the specific case of LSIL, we found that association with MMP-9 is more evident when there is the simultaneous presence of an infectious agent. Thus, the expression of MMP-9 in cervical smears increases according to the grade of cervical lesion and LSIL in the presence of infectious agents showed higher MMP-9 expression than women with LSIL without infectious agents.

  14. Increased Expression of Angiopoietins and Tie2 in the Lungs of Chronic Asthmatic Mice

    PubMed Central

    Makinde, Toluwalope O.; Agrawal, Devendra K.

    2011-01-01

    Angiopoietin (Ang)1 and Ang2 are ligands for Tie2 tyrosine kinase receptor (Tie2). Elevated levels of Ang1 and Ang2 in induced sputum of patients with asthma have been reported, with a positive correlation of Ang2 levels with the severity of airway occlusion. Although studies have shown Tie2-mediated regulation of nonvascular cells in some pathological conditions, current knowledge on Tie2 signaling in asthma is limited to the vasculature. We examined the expression pattern of Ang1, Ang2, vascular endothelial growth factor (VEGF), and Tie2 and their correlation with the degree of airway remodeling in the lung of ovalbumin (OVA)-sensitized and OVA-challenged mice with airway hyperresponsiveness. Lung tissues were isolated from Balb/c mice after OVA sensitization and challenge. Hematoxylin and eosin, periodic acid-Schiff, and trichrome staining were used to show the lung pathology. The expression of Ang1, Ang2, VEGF, and Tie2 was examined using immunofluorescence, Western blot, ELISA, and real-time PCR. In the lung of normal mice, Tie2 expression was detected only in the blood vessels. However, in the lung of OVA-sensitized and OVA-challenged mice, Tie2 was abundantly expressed in airway epithelial cells and in a subset of macrophages in addition to constitutive expression in pulmonary vessels. The increase in Tie2 expression correlated with the severity of airway remodeling. Macrophages and airway epithelial cells express Ang2 and VEGF only in allergic models. Ang1 was constitutively expressed, with a decrease in mRNA level in allergic models. In conclusion, increased expression of Tie2 and Ang2 in allergic airway epithelium and alveolar macrophages correlates with the severity of airway remodeling. PMID:20463289

  15. Glial cell line-derived neurotrophic factor (GDNF) expression and NMJ plasticity in skeletal muscle following endurance exercise.

    PubMed

    Gyorkos, A M; McCullough, M J; Spitsbergen, J M

    2014-01-17

    Glial cell line-derived neurotrophic factor (GDNF) supports and maintains the neuromuscular system during development and through adulthood by promoting neuroplasticity. The aim of this study was to determine if different modes of exercise can promote changes in GDNF expression and neuromuscular junction (NMJ) morphology in slow- and fast-twitch muscles. Rats were randomly assigned to a run training (run group), swim training (swim group), or sedentary control group. GDNF protein content was determined by enzyme-linked immunosorbant assay. GDNF protein content increased significantly in soleus (SOL) following both training protocols (P<0.05). Although not significant, an increase of 60% in the extensor digitorum longus (EDL) followed swim-training (NS; P<0.06). NMJ morphology was analyzed by measuring α-bungarotoxin labeled post-synaptic end plates. GDNF content and total end plate area were positively correlated. End plate area decreased in EDL of the run group and increased in SOL of the swim group. The results indicate that GDNF expression and NMJ morphological changes are activity dependent and that different changes may be observed by varying the exercise intensity in slow- and fast-twitch fibers.

  16. Expression Profiling of Glucosinolate Biosynthetic Genes in Brassica oleracea L. var. capitata Inbred Lines Reveals Their Association with Glucosinolate Content.

    PubMed

    Robin, Arif Hasan Khan; Yi, Go-Eun; Laila, Rawnak; Yang, Kiwoung; Park, Jong-In; Kim, Hye Ran; Nou, Ill-Sup

    2016-01-01

    Glucosinolates are the biochemical compounds that provide defense to plants against pathogens and herbivores. In this study, the relative expression level of 48 glucosinolate biosynthesis genes was explored in four morphologically-different cabbage inbred lines by qPCR analysis. The content of aliphatic and indolic glucosinolate molecules present in those cabbage lines was also estimated by HPLC analysis. The possible association between glucosinolate accumulation and related gene expression level was explored by principal component analysis (PCA). The genotype-dependent variation in the relative expression level of different aliphatic and indolic glucosinolate biosynthesis genes is the novel result of this study. A total of eight different types of glucosinolates, including five aliphatic and three indolic glucosinolates, was detected in four cabbage lines. Three inbred lines BN3383, BN4059 and BN4072 had no glucoraphanin, sinigrin and gluconapin detected, but the inbred line BN3273 had these three aliphatic glucosinolate compounds. PCA revealed that a higher expression level of ST5b genes and lower expression of GSL-OH was associated with the accumulation of these three aliphatic glucosinolate compounds. PCA further revealed that comparatively higher accumulation of neoglucobrassicin in the inbred line, BN4072, was associated with a high level of expression of MYB34 (Bol017062) and CYP81F1 genes. The Dof1 and IQD1 genes probably trans-activated the genes related to biosynthesis of glucoerucin and methoxyglucobrassicin for their comparatively higher accumulation in the BN4059 and BN4072 lines compared to the other two lines, BN3273 and BN3383. A comparatively higher progoitrin level in BN3273 was probably associated with the higher expression level of the GSL-OH gene. The cabbage inbred line BN3383 accounted for the significantly higher relative expression level for the 12 genes out of 48, but this line had comparatively lower total glucosinolates detected

  17. Organophosphate pesticides increase the expression of alpha glutathione S-transferase in HepG2 cells.

    PubMed

    Medina-Díaz, I M; Rubio-Ortíz, M; Martínez-Guzmán, M C; Dávalos-Ibarra, R L; Rojas-García, A E; Robledo-Marenco, M L; Barrón-Vivanco, B S; Girón-Pérez, M I; Elizondo, G

    2011-12-01

    Chlorpyrifos and methyl parathion are among the most widely used insecticides in the world. Human populations are constantly exposed to low doses of both due to their extensive use and presence in food and drinking water. Glutathione S-transferase (GST) catalyzes the conjugation of glutathione on electrophilic substrates and is an important line of defense in the protection of cellular components from reactive species. GST alpha1 (GSTA1) is the predominant isoform of GST expressed in the human liver; thus, determining the effect of insecticides on GSTA1 transcription is very important. In the present study, we analyzed the effects of methyl parathion and chlorpyrifos on GSTA1 gene expression in HepG2 cells using real time PCR, and activity and immunoreactive protein assays. The results demonstrated that exposure to methyl parathion and chlorpyrifos increased the level of GSTA1 mRNA, GSTA1 immunoreactive protein and GST activity relative to a control. These results demonstrated that these insecticides can increase the expression of GSTA1. In conclusion, HepG2 cell cultures treated with methyl parathion and chlorpyrifos could be a useful model for studying the function of GSTA1 and its role in the metabolism of xenobiotics in the liver. PMID:21907274

  18. Expression of mammalian O6-alkylguanine-DNA alkyltransferase in a cell line sensitive to alkylating agents.

    PubMed

    Dolan, M E; Norbeck, L; Clyde, C; Hora, N K; Erickson, L C; Pegg, A E

    1989-09-01

    Chinese hamster ovary cells (CHO) were co-transfected with pSV2neo and sheared DNA from either a human cell line (HT29) expressing high levels of O6-alkylguanine-DNA alkyltransferase (AGT) or from a cell line (BE) deficient in this activity. Cells expressing the selectable marker were obtained by exposure to G418 and colonies resistant to alkylation damage isolated by growth in the presence of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). The number of colonies of cells expressing AGT activity arising after transfection with DNA from BE cells was similar to the number arising from cells exposed to HT29 DNA. Although the amount of AGT repair protein expressed in the transfectant colonies from this experiment was relatively low, these results indicate that repair of alkylation damage can be restored in AGT-deficient cells by transfection of human DNA from both repair-deficient and proficient cells. A separate transfection of CHOMG cells [a mutant of CHO cells resistant to the drug, methylglyoxal bis(guanylhydrazone) (MGBG)] with HT29 DNA and pSV2neo followed by selection of G418 and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in three colonies with high AGT levels. These transfectants had different growth rates and expressed levels of the AGT protein between 230 and 300 fmol/mg protein. The transfectants were as resistant to the cytotoxic effects of BCNU, Clomesone, methylnitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) as HT29 cells which were much more resistant than the parental CHOMG cells. Pretreatment of transfectant cells with 0.4 mM O6-methylguanine for 24 h reduced AGT activity to 14% basal levels, which upon removal of the base increased to approximately 74% basal level within 8 h. The sensitivity to the cytotoxic effects of both the chloroethylating and methylating agents was enhanced by treatment with O6-methylguanine. In the same manner, the number of BCNU-induced DNA interstrand cross-links increased in transfectant

  19. Small Businesses Save Big: A Borrower's Guide To Increase the Bottom Line Using Energy Efficiency (Fact Sheet)

    SciTech Connect

    Not Available

    2015-01-01

    Dollars saved through energy efficiency can directly impact your bottom line. Whether you are planning for a major renovation or upgrading individual pieces of building equipment, these improvements can help reduce operating costs, save on utility bills, and boost profits. This fact sheet provides a guide for small businesses to find the resources to increase the energy efficiency of their buildings.

  20. Increasing Student Interest and Comprehension of Production Planning and Control and Operations Performance Measurement Concepts Using a Production Line Game

    ERIC Educational Resources Information Center

    Cox, James F., III; Walker, Edward D., II

    2005-01-01

    Production planning and control (PPC) systems and operations performance measures are topics that students generally find both boring and difficult to understand. In the article, the authors present a production line game that they have found to be an effective tool to increase student interest in the topics as well as student comprehension. The…

  1. SV40-IMMORTALIZED NON-TUMORIGENIC AND TUMORIGENIC CELL LINES DIFFER IN EXPRESSION OF HALLMARK VIRAL RESPONSE MRNAS

    EPA Science Inventory

    SV40-Immortalized Non-Tumorigenic and Tumorigenic Cell Lines Differ in Expression of Hallmark Viral Response mRNAs.

    Prior to the use of an in vitra/in viva transformation system to examine the tumorigenic activity of environmental contaminants, in vitra gene expression pa...

  2. Electroporation transiently decreases GJB2 (connexin 26) expression in B16/BL6 melanoma cell line.

    PubMed

    Rangel, Marcelo Monte Mór; Chaible, Lucas Martins; Nagamine, Marcia Kazumi; Mennecier, Gregory; Cogliati, Bruno; de Oliveira, Krishna Duro; Fukumasu, Heidge; Sinhorini, Idércio Luiz; Mir, Lluis Maria; Dagli, Maria Lúcia Zaidan

    2015-02-01

    Connexins are proteins that form gap junctions. Perturbations in the cell membrane reportedly promote changes in the expression profile of connexins. Electroporation promotes destabilization by applying electrical pulses, and this procedure is used in electrochemotherapy and gene therapy, among others. This in vitro work aimed to study the interference of electroporation on the expression profile of GJB2 (Cx26 gene) and Connexin 26 in melanoma cell line B16/BL6. The techniques of immunocytochemistry, Western blot, and real-time PCR were used. After electroporation, cells showed a transient decrease in GJB2 mRNA. The immunostaining of Cx26 showed no noticeable change after electroporation at different time points. However, Western blot showed a significant reduction in Cx26 30 min after electroporation. Our results showed that electroporation interferes transiently in the expression of Connexin 26 in melanoma and are consistent with the idea that electroporation is a process of intense stress that promotes cell homeostatic imbalance and results in disruption of cell physiological processes such as transcription and translation.

  3. Bortezomib and Arsenic Trioxide Activity on a Myelodysplastic Cell Line (P39): A Gene Expression Study

    PubMed Central

    Savlı, Hakan; Galimberti, Sara; Sünnetçi, Deniz; Canestraro, Martina; Palumbo, Giuseppe; Nagy, Balint; Raimondo, Francesco Di; Petrini, Mario

    2015-01-01

    Objective: We aimed to understand the molecular pathways affected by bortezomib and arsenic trioxide treatment on myelomonocytoid cell line P39. Materials and Methods: Oligonucleotide microarray platforms were used for gene expression and pathway analysis. Confirmation studies were performed using quantitative real time PCR. Results: Bortezomib treatment has shown upregulated DIABLO and NF-κBIB (a NF-κB inhibitor) and downregulated NF-κB1, NF-κB2, and BIRC1 gene expressions. Combination treatment of the two compounds showed gene expression deregulations in concordance by the results of single bortezomib treatment. Especially, P53 was a pathway more significantly modified and a gene network centralized around the beta estradiol gene. Beta estradiol, BRCA2, and FOXA1 genes were remarkable deregulations in our findings. Conclusion: Results support the suggestions about possible use of proteasome inhibitors in the treatment of high-risk myelodysplastic syndrome (MDS). NF-κB was observed as an important modulator in leukemic transformation of MDS. PMID:25913414

  4. Expression pattern of the apoptosis-stimulating protein of p53 family in p53+ human breast cancer cell lines

    PubMed Central

    2013-01-01

    Background The apoptosis-stimulating protein of p53 (ASPP) family comprises three members, namely, ASPP1, ASPP2, and iASPP. They regulate the promotive effect of p53 on apoptosis. Breast cancer (BC) remains as one of the leading causes of cancer or cancer-related mortality among women. However, the relationship between the ASPP family members and p53, as well as the dissemination and expression pattern of ASPP family members in p53+ BC, has not been elucidated. Our objectives are to detect the expression of ASPP family members in p53+ BC cell lines and determine its significance in tumor cell apoptosis. Methods The mRNA expression of ASPP family members in five p53+ BC cell lines was detected through RT-PCR and assayed using Quality-one software. The p53 protein expression was detected by immunohistochemistry. Afterward, the apoptosis indices of the five BC cell lines were detected by flow cytometry. Results The iASPP mRNA was expressed in Bcap-37, MCF-7, and HBL-100. Compared with the human peripheral blood mononuclear cells, significant differences were found in the ASPP1 mRNA in Bcap-37, MDA-MB-231, MCF-7, and HBL-100 (p < 0.05), except that in ZR-75-30 (p > 0.05). The ASPP2 mRNA was expressed in MDA-MB-231, Bcap-37, and MCF-7, but not in HBL-100 and ZR-75-30. The p53 protein was expressed in five breast cancer cell lines. ZR-75-30 and MDA-MB-231 apoptosis indices were higher than those of other breast cancer cell line and peripheral blood mononuclear cells (p < 0.01). Conclusions The mRNA expression of ASPP family members varied in the five p53+ BC cell lines. The results also verified that the family members have an important function in apoptosis, which was promoted by p53 protein. ZR-75-30 BC showed high apoptosis index, without expression of any ASPP family members, indicating that the pathway of apoptosis in this cell line may be related to other cell transduction pathway. MDA-MB-231, Bcap37, and MCF-7 cell lines all expressed ASPP1/2. However, the

  5. SPARCL1 Expression Increases With Preoperative Radiation Therapy and Predicts Better Survival in Rectal Cancer Patients

    SciTech Connect

    Kotti, Angeliki Holmqvist, Annica; Albertsson, Maria; Sun, Xiao-Feng

    2014-04-01

    Purpose: The secreted protein acidic and rich in cysteine-like 1 (SPARCL1) is expressed in various normal tissues and many types of cancers. The function of SPARCL1 and its relationship to a patient's prognosis have been studied, whereas its relationship to radiation therapy (RT) is not known. Our aim was to investigate the expression of SPARCL1 in rectal cancer patients who participated in a clinical trial of preoperative RT. Methods and Materials: The study included 136 rectal cancer patients who were randomized to undergo preoperative RT and surgery (n=63) or surgery alone (n=73). The expression levels of SPARCL1 in normal mucosa (n=29), primary tumor (n=136), and lymph node metastasis (n=35) were determined by immunohistochemistry. Results: Tumors with RT had stronger SPARCL1 expression than tumors without RT (P=.003). In the RT group, strong SPARCL1 expression was related to better survival than weak expression in patients with stage III tumors, independent of sex, age, differentiation, and margin status (P=.022; RR = 18.128; 95% confidence interval, 1.512-217.413). No such relationship was found in the non-RT group (P=.224). Further analysis of interactions among SPARCL1 expression, RT, and survival showed statistical significance (P=.024). In patients with metastases who received RT, strong SPARCL1 expression was related to better survival compared to weak expression (P=.041) but not in the non-RT group (P=.569). Conclusions: SPARCL1 expression increases with RT and is related to better prognosis in rectal cancer patients with RT but not in patients without RT. This result may help us to select the patients best suited for preoperative RT.

  6. Reduced glycosylation of human cell lines increases susceptibility to CD4-independent infection by human immunodeficiency virus type 2 (LAV-2/B).

    PubMed Central

    Talbot, S J; Weiss, R A; Schulz, T F

    1995-01-01

    The human immunodeficiency virus type 2 (HIV-2) strain LAV-2/B is able to infect a variety of human cell lines via a CD4-independent pathway. We have used the glycosylation inhibitors tunicamycin, swainsonine, and deoxymannojirimycin to further characterize this putative alternative receptor for HIV-2 (LAV-2/B). These antibiotics resulted in an increase (5- to 30-fold) in the susceptibility of a variety of CD4- human cell lines to infection by LAV-2/B (RD, HeLa, HT29, Rsb, Heb7a, Hos, and Daudi). Several nonprimate cell lines (mink Mv-1-lu, rabbit SIRC, hamster a23, mouse NIH 3T3, cat CCC, and rat HSN) remained resistant to infection by LAV-2/B after treatment with glycosylation inhibitors, suggesting that they do not express the HIV-2 CD4-independent receptor. Two of these nonprimate cell lines are readily infected by HIV-2 when they express CD4 (Mv-1-lu and CCC). Treatment of human cells with neuraminidase had no effect on subsequent infection by LAV-2/B, suggesting that the increase in susceptibility to infection of deglycosylated cells is not due to a change in the electrostatic charge of the cell surface. Treatment of RD CD4- cells and HeLa CD4+ cells with a variety of proteases resulted in a 75 to 90% decrease in infection by LAV-2/B when compared with untreated cells. Taken together, all these data suggest that HIV-2 can utilize a membrane glycoprotein other than CD4 to attach and fuse with a variety of human cells. PMID:7745686

  7. The transposition frequency of Tag1 elements is increased in transgenic Arabidopsis lines.

    PubMed Central

    Bhatt, A M; Lister, C; Crawford, N; Dean, C

    1998-01-01

    Tag1 was identified as a highly active endogenous transposable element in transgenic Arabidopsis thaliana Landsberg erecta plants carrying the maize transposable element Activator (Ac). Here, we describe experiments designed to determine the basis for the high activity of Tag1. The frequency of transposition of Tag1 elements was compared in lines containing or lacking Ac transposase to assess the effect of Ac transposase on Tag1 activity. Three populations of nontransgenic plants, including nontransformed regenerants, were also analyzed. The high level of activity of Tag1 did not correlate with the presence or absence of Ac transposase but was significantly higher in transgenic lines. This result was maintained through at least six generations after transformation. These data suggest that Tag1 transposition is stimulated by processes that occur during the Agrobacterium transformation and that thereafter remain active. Two Tag1 elements are tightly linked in the Landsberg erecta genome and map to the lower arm of chromosome 1. Tag1 elements were found in only a few A. thaliana ecotypes but were present in four other Arabidopsis species. PMID:9501115

  8. Increased CD44 gene expression in lymphocytes derived from Alzheimer disease patients.

    PubMed

    Uberti, D; Cenini, G; Bonini, S A; Barcikowska, M; Styczynska, M; Szybinska, A; Memo, M

    2010-01-01

    In this study, we demonstrated for the first time an increased CD44 gene expression in lymphocytes derived from Alzheimer's disease (AD) patients in comparison with healthy subjects. CD44 is a surface antigen expressed by cells of the immune and central nervous system as well as in a variety of other tissues. Functioning as adhesion molecule, CD44 is furthermore involved in driving immune response into infected tissues, including the CNS. We also found that lymphocytes of the same patients expressed significant levels of unfolded p53 isoform, confirming what we already demonstrated in fibroblasts and lymphocytes derived from other cohorts of AD patients. A correlation between p53 and CD44 expression has been well demonstrated in cancer cells, suggesting that CD44 could be a target gene of mutant p53, or either mutant p53 could lack its ability to negatively regulate CD44 expression. The contemporaneous increased expression of unfolded p53 and CD44 in AD lymphocytes may suggest that these two molecules cross-talk together participating in peripheral immune response during the development of the disease.

  9. Varicella-zoster viruses associated with post-herpetic neuralgia induce sodium current density increases in the ND7-23 Nav-1.8 neuroblastoma cell line.

    PubMed

    Kennedy, Peter G E; Montague, Paul; Scott, Fiona; Grinfeld, Esther; Ashrafi, G H; Breuer, Judith; Rowan, Edward G

    2013-01-01

    Post-herpetic neuralgia (PHN) is the most significant complication of herpes zoster caused by reactivation of latent Varicella-Zoster virus (VZV). We undertook a heterologous infection in vitro study to determine whether PHN-associated VZV isolates induce changes in sodium ion channel currents known to be associated with neuropathic pain. Twenty VZV isolates were studied blind from 11 PHN and 9 non-PHN subjects. Viruses were propagated in the MeWo cell line from which cell-free virus was harvested and applied to the ND7/23-Nav1.8 rat DRG x mouse neuroblastoma hybrid cell line which showed constitutive expression of the exogenous Nav 1.8, and endogenous expression of Nav 1.6 and Nav 1.7 genes all encoding sodium ion channels the dysregulation of which is associated with a range of neuropathic pain syndromes. After 72 hrs all three classes of VZV gene transcripts were detected in the absence of infectious virus. Single cell sodium ion channel recording was performed after 72 hr by voltage-clamping. PHN-associated VZV significantly increased sodium current amplitude in the cell line when compared with non-PHN VZV, wild-type (Dumas) or vaccine VZV strains ((POka, Merck and GSK). These sodium current increases were unaffected by acyclovir pre-treatment but were abolished by exposure to Tetrodotoxin (TTX) which blocks the TTX-sensitive fast Nav 1.6 and Nav 1.7 channels but not the TTX-resistant slow Nav 1.8 channel. PHN-associated VZV sodium current increases were therefore mediated in part by the Nav 1.6 and Nav 1.7 sodium ion channels. An additional observation was a modest increase in message levels of both Nav1.6 and Nav1.7 mRNA but not Nav 1.8 in PHN virally infected cells. PMID:23382806

  10. Varicella-Zoster Viruses Associated with Post-Herpetic Neuralgia Induce Sodium Current Density Increases in the ND7-23 Nav-1.8 Neuroblastoma Cell Line

    PubMed Central

    Kennedy, Peter G. E.; Montague, Paul; Scott, Fiona; Grinfeld, Esther; Ashrafi, G. H.; Breuer, Judith; Rowan, Edward G.

    2013-01-01

    Post-herpetic neuralgia (PHN) is the most significant complication of herpes zoster caused by reactivation of latent Varicella-Zoster virus (VZV). We undertook a heterologous infection in vitro study to determine whether PHN-associated VZV isolates induce changes in sodium ion channel currents known to be associated with neuropathic pain. Twenty VZV isolates were studied blind from 11 PHN and 9 non-PHN subjects. Viruses were propagated in the MeWo cell line from which cell-free virus was harvested and applied to the ND7/23-Nav1.8 rat DRG x mouse neuroblastoma hybrid cell line which showed constitutive expression of the exogenous Nav 1.8, and endogenous expression of Nav 1.6 and Nav 1.7 genes all encoding sodium ion channels the dysregulation of which is associated with a range of neuropathic pain syndromes. After 72 hrs all three classes of VZV gene transcripts were detected in the absence of infectious virus. Single cell sodium ion channel recording was performed after 72 hr by voltage-clamping. PHN-associated VZV significantly increased sodium current amplitude in the cell line when compared with non-PHN VZV, wild-type (Dumas) or vaccine VZV strains ((POka, Merck and GSK). These sodium current increases were unaffected by acyclovir pre-treatment but were abolished by exposure to Tetrodotoxin (TTX) which blocks the TTX-sensitive fast Nav 1.6 and Nav 1.7 channels but not the TTX-resistant slow Nav 1.8 channel. PHN-associated VZV sodium current increases were therefore mediated in part by the Nav 1.6 and Nav 1.7 sodium ion channels. An additional observation was a modest increase in message levels of both Nav1.6 and Nav1.7 mRNA but not Nav 1.8 in PHN virally infected cells. PMID:23382806

  11. Effect of boswellia thurifera gum methanol extract on cytotoxicity and p53 gene expression in human breast cancer cell line.

    PubMed

    Yazdanpanahi, Nasrin; Behbahani, Mandana; Yektaeian, Afsaneh

    2014-01-01

    Boswellia has been widely used in traditional medicine for the treatment of different diseases such as cancer in Iran. The aim of this study was to evaluate the effect of the gum methanol extract of Boswellia thurifera on the viability and P53 gene expression of cultured breast cancer cells. The gum methanol extract was obtained in various concentrations using the maceration method. Normal (HEK-293) and cancer (MDA-MB-231) human cells were cultured and treated with various concentrations of the extract. Then MTT assay was used for the study of cytotoxic effect of the extract and real time PCR method was also applied for the investigation of P53 gene expression in cancer cells. The IC50 of the extract against cancer cells was 80 µg/mL and had less cytotoxic effect in normal cells. The effect of the extract was dose dependent. Induction of P53 expression by extract was also significantly more in treated cancer cells than untreated cells. This inductive effect in cells was higher after 12 h treatment than it was after 6 h. The results of the current study show that gum methanol extract of Boswellia thurifera has probably anti-cancer effects and could induce P53 gene transcription and toxicity in the cultured breast cancer cell line. The increase of P53 gene specific mRNA may be a mechanism of gum methanol extract induced cytotoxicity. However, for a definitive conclusion, further studies on other cell lines as well as animal models and subsequent clinical studies are warranted. PMID:25237368

  12. Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line.

    PubMed

    Poklepovich, Tomas J; Urtasun, Nicolás; Miranda, María V; Nusblat, Alejandro D; Nudel, Clara B

    2015-04-01

    Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol) with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzyme DES7 is encoded by the gene TTHERM_00310640, identified with RNAi and gene knock-out experiments, but has not yet been heterologously expressed actively in any organism. A model derived from its amino acid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The protein has catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in the synthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity, we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line, identified and measured the steroid metabolites formed, and extended the actual knowledge on its localization. We found that the accumulation of 7-dehydrocholesterol could be increased 16-40-fold in Spodopterafrugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7. The protein was detected in the microsomal fraction, in accordance with previous reports. Although the electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown, we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite of the large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that there is significant functional conservation of the electron donors, since the ciliate's sterol desaturase can function in the context of the insect electron transport system. The results achieved demonstrate that DES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insect cell line.

  13. Examining the Relationship Between Cu-ATSM Hypoxia Selectivity and Fatty Acid Synthase Expression in Human Prostate Cancer Cell Lines

    PubMed Central

    Vāvere, Amy L.; Lewis, Jason S.

    2013-01-01

    Introduction PET imaging with Cu-ATSM for delineating hypoxia has provided valuable clinical information, but investigations in animal models of prostate cancer have shown some inconsistencies. As a defense mechanism in prostate cancer cells, the fatty acid synthesis pathway harnesses its oxidizing power for improving the redox balance despite conditions of extreme hypoxia, potentially altering Cu-ATSM hypoxia-selectivity. Methods Human prostate tumor cultured cell lines (PC-3, 22Rv1, LNCaP, and LAPC-4), were treated with an FAS inhibitor (C75, 100 μM) under anoxia. 64Cu-ATSM uptake into these treated cells, and non-treated anoxic cells, was then examined. Fatty acid synthase (FAS) expression level in each cell line was subsequently quantified by ELISA. An additional study was performed in PC-3 cells to examine the relationship between the restoration of 64Cu-ATSM hypoxia-selectivity and the concentration of C75 (100, 20, 4, or 0.8 μM) administered to the cells. Results Inhibition of fatty acid synthesis with C75 resulted in a significant increase in 64Cu-ATSM retention into prostate tumor cells in vitro under anoxia over 60 mins. Inhibition studies demonstrated higher uptake values of 20.9 ± 3.27, 103.0 ± 32.6, 144.2 ± 32.3, and 200.1 ± 79.3% at 15 mins over control values for LAPC-4, PC-3, LNCaP, and 22Rv1 cells, respectively. A correlation was seen (R2 = 0.911) with FAS expression plotted against % change in 64Cu-ATSM uptake with C75 treatment. Conclusions Although Cu-ATSM has clinical relevance in the PET imaging of hypoxia in many tumor types, its translation to the imaging of prostate cancer may be limited by the over-expression of FAS associated with prostatic malignancies. PMID:18355682

  14. Effect of Boswellia Thurifera Gum Methanol Extract on Cytotoxicity and P53 Gene Expression in Human Breast Cancer Cell Line

    PubMed Central

    Yazdanpanahi, Nasrin; Behbahani, Mandana; Yektaeian, Afsaneh

    2014-01-01

    Boswellia has been widely used in traditional medicine for the treatment of different diseases such as cancer in Iran. The aim of this study was to evaluate the effect of the gum methanol extract of Boswellia thurifera on the viability and P53 gene expression of cultured breast cancer cells. The gum methanol extract was obtained in various concentrations using the maceration method. Normal (HEK-293) and cancer (MDA-MB-231) human cells were cultured and treated with various concentrations of the extract. Then MTT assay was used for the study of cytotoxic effect of the extract and real time PCR method was also applied for the investigation of P53 gene expression in cancer cells. The IC50 of the extract against cancer cells was 80 µg/mL and had less cytotoxic effect in normal cells. The effect of the extract was dose dependent. Induction of P53 expression by extract was also significantly more in treated cancer cells than untreated cells. This inductive effect in cells was higher after 12 h treatment than it was after 6 h. The results of the current study show that gum methanol extract of Boswellia thurifera has probably anti-cancer effects and could induce P53 gene transcription and toxicity in the cultured breast cancer cell line. The increase of P53 gene specific mRNA may be a mechanism of gum methanol extract induced cytotoxicity. However, for a definitive conclusion, further studies on other cell lines as well as animal models and subsequent clinical studies are warranted. PMID:25237368

  15. Heat Stress and Lipopolysaccharide Stimulation of Chicken Macrophage-Like Cell Line Activates Expression of Distinct Sets of Genes

    PubMed Central

    Slawinska, Anna; Hsieh, John C.; Schmidt, Carl J.; Lamont, Susan J.

    2016-01-01

    Acute heat stress requires immediate adjustment of the stressed individual to sudden changes of ambient temperatures. Chickens are particularly sensitive to heat stress due to development of insufficient physiological mechanisms to mitigate its effects. One of the symptoms of heat stress is endotoxemia that results from release of the lipopolysaccharide (LPS) from the guts. Heat-related cytotoxicity is mitigated by the innate immune system, which is comprised mostly of phagocytic cells such as monocytes and macrophages. The objective of this study was to analyze the molecular responses of the chicken macrophage-like HD11 cell line to combined heat stress and lipopolysaccharide treatment in vitro. The cells were heat-stressed and then allowed a temperature-recovery period, during which the gene expression was investigated. LPS was added to the cells to mimic the heat-stress-related endotoxemia. Semi high-throughput gene expression analysis was used to study a gene panel comprised of heat shock proteins, stress-related genes, signaling molecules and immune response genes. HD11 cell line responded to heat stress with increased mRNA abundance of the HSP25, HSPA2 and HSPH1 chaperones as well as DNAJA4 and DNAJB6 co-chaperones. The anti-apoptotic gene BAG3 was also highly up-regulated, providing evidence that the cells expressed pro-survival processes. The immune response of the HD11 cell line to LPS in the heat stress environment (up-regulation of CCL4, CCL5, IL1B, IL8 and iNOS) was higher than in thermoneutral conditions. However, the peak in the transcriptional regulation of the immune genes was after two hours of temperature-recovery. Therefore, we propose the potential influence of the extracellular heat shock proteins not only in mitigating effects of abiotic stress but also in triggering the higher level of the immune responses. Finally, use of correlation networks for the data analysis aided in discovering subtle differences in the gene expression (i.e. the role

  16. Changes in Protein Expression in Two Cholangiocarcinoma Cell Lines Undergoing Formation of Multicellular Tumor Spheroids In Vitro

    PubMed Central

    Mischiati, Carlo; Ura, Blendi; Roncoroni, Leda; Elli, Luca; Cervellati, Carlo; Squerzanti, Monica; Conte, Dario; Doneda, Luisa; Polverino de Laureto, Patrizia; de Franceschi, Giorgia; Calza, Roberta; Barrero, Carlos A.; Merali, Salim; Ferrari, Carlo; Bergamini, Carlo M.; Agostinelli, Enzo

    2015-01-01

    Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and resistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutaminase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The arising protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conversion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the carbohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen. PMID:25756965

  17. WISP-1 increases MMP-2 expression and cell motility in human chondrosarcoma cells.

    PubMed

    Hou, Chun-Han; Chiang, Yi-Chun; Fong, Yi-Chin; Tang, Chih-Hsin

    2011-06-01

    Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matricellular proteins. However, the effect of WISP-1 on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that WISP-1 increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells (JJ012 cells). We also found that human chondrosarcoma tissues had significant expression of the WISP-1 which was higher than that in normal cartilage. α5β1 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) inhibited the WISP-1-induced increase of the migration and MMP-2 up-regulation of chondrosarcoma cells. WISP-1 stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors also suppressed the cell migration and MMP-2 expression enhanced by WISP-1. Moreover, WISP-1 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-2 promoter. Taken together, our results indicated that WISP-1 enhances the migration of chondrosarcoma cells by increasing MMP-2 expression through the α5β1 integrin receptor, FAK, MEK, ERK, p65 and NF-κB signal transduction pathway.

  18. Increase of Calcium Sensing Receptor Expression Is Related to Compensatory Insulin Secretion during Aging in Mice

    PubMed Central

    Oh, Yoon Sin; Seo, Eun-Hui; Lee, Young-Sun; Cho, Sung Chun; Jung, Hye Seung; Park, Sang Chul; Jun, Hee-Sook

    2016-01-01

    Type 2 diabetes is caused by both insulin resistance and relative insulin deficiency. To investigate age-related changes in glucose metabolism and development of type 2 diabetes, we compared glucose homeostasis in different groups of C57BL/6J mice ranging in age from 4 months to 20 months (4, 8, 12, 16 and 20 months). Interestingly, we observed that non-fasting glucose levels were not significantly changed, but glucose tolerance gradually increased by 20 months of age, whereas insulin sensitivity declined with age. We found that the size of islets and glucose-stimulated insulin secretion increased with aging. However, mRNA expression of pancreatic and duodenal homeobox 1 and granuphilin was decreased in islets of older mice compared with that of 4-month-old mice. Serum calcium (Ca2+) levels were significantly decreased at 12, 20 and 28 months of age compared with 4 months and calcium sensing receptor (CaSR) mRNA expression in the islets significantly increased with age. An extracellular calcium depletion agent upregulated CaSR mRNA expression and consequently enhanced insulin secretion in INS-1 cells and mouse islets. In conclusion, we suggest that decreased Ca2+ levels and increased CaSR expression might be involved in increased insulin secretion to compensate for insulin resistance in aged mice. PMID:27441644

  19. Constitutive S-adenosylmethionine decarboxylase gene expression increases drought tolerance through inhibition of reactive oxygen species accumulation in Arabidopsis.

    PubMed

    Wi, Soo Jin; Kim, Soo Jin; Kim, Woo Taek; Park, Ky Young

    2014-05-01

    Using subtractive hybridization analysis, the S-adenosylmethionine decarboxylase (SAMDC) gene from Capsicum annuum was isolated and renamed CaSAMDC. We generated independent transgenic Arabidopsis (Arabidopsis thaliana) lines constitutively expressing a 35S::CaSAMDC construct. Drought tolerance was significantly enhanced in Arabidopsis T4 transgenic homozygous lines as compared to wild-type (WT) plants. The levels of main polyamines (PAs) were more significantly increased in CaSAMDC-overexpressing transgenic plants after 6 h of drought stress as compared to stressed WT plants. Basal transcription of polyamine oxidase (PAO) showed at a much higher level in unstressed-transgenic plants as compared to unstressed WT plants. However, the difference in PAO transcription level between WT and transgenic plants was reduced after drought stress. Cellular accumulation of reactive oxygen species (ROS) was significantly reduced following drought stress in transgenic Arabidopsis plants as compared to WT plants. These results were in agreement with additional observations that stress-induced ROS generation, as determined by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), was significantly suppressed while transcription of ROS-detoxifying enzymes was notably elevated in transgenic lines in response to drought stress. Further, ROS-induced transcription of the metacaspase II gene was remarkably inhibited in transgenic plants. Collectively, these results suggest that drought stress tolerance due to reduction of ROS production and enhancement of ROS detoxification can be attributed to elevation of PAs.

  20. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

    PubMed Central

    Terrón-González, L.; Medina, C.; Limón-Mortés, M. C.; Santero, E.

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance. PMID:23346364

  1. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

    PubMed

    Terrón-González, L; Medina, C; Limón-Mortés, M C; Santero, E

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

  2. Natalizumab treatment leads to an increase in circulating CXCR3-expressing B cells

    PubMed Central

    Penttilä, Tarja-Leena; Airas, Laura

    2016-01-01

    Objective: To study the effects of natalizumab treatment on subgroups of circulating peripheral blood B cell populations. Methods: We studied the proportions and absolute numbers of CD19+CD20+, CD10+, and CD5+ B cell populations, and determined very late activation antigen-4 and chemokine receptor CXCR3, CCR5, and CCR6 expression on B cells in the peripheral blood of 14 natalizumab-treated patients with relapsing-remitting multiple sclerosis. Five blood samples per patient were obtained longitudinally before and during the first year of treatment. Blood samples were analyzed by 6-color flow cytometry. Results: Proportions of B cells and CD10+ pre–B cells were significantly increased, and very late activation antigen-4 expression on the B cell surface was significantly decreased already after 1 week of natalizumab treatment. Natalizumab-induced sustained increase in the proportion and absolute number of CXCR3-expressing B cells was statistically significant after 1 month of treatment. There were no changes in the proportions of CCR5- or CCR6-expressing B cells. Conclusions: The rapid and persistent increase in circulating CXCR3-expressing B cells in response to natalizumab treatment possibly reflects the relevance of this chemokine receptor in controlling migration of B cells into the CNS in humans in vivo. PMID:27800533

  3. Decrease in PTEN and increase in Akt expression and neuron size in aged rat spinal cord

    PubMed Central

    Rodrigues De Amorim, Miguel Augusto; Garcia-Segura, Luis Miguel; Goya, Rodolfo Gustavo; Portiansky, Enrique Leo

    2010-01-01

    PTEN is a tumor suppressor gene known to play an important role in the regulation of cell size. In this study we compared PTEN expression in the spinal cord of young (5 mo.) versus aged (32 mo.) female rats and correlated them with alterations in neuron size and morphology in the same animals. Total and phosphorylated PTEN (pPTEN) as well as its downstream target phosphorylated Akt (pAkt) were assessed by western blotting. Spinal cord neurons were morphometrically characterized. Total PTEN, pPTEN and total Akt expression were significantly higher in young rats than in aged animals. Expression of pAkt was stronger in aged animals. A significant increase in neuronal size was observed in large motoneurons of aged as compared with young rats. Our data show that in the spinal cord of rats, neuronal PTEN expression diminishes with advanced age while neuronal size increases. These results suggest that in the spinal cord, an age-related reduction in PTEN and increase of pAkt expression may be involved in the progressive enlargement of neurons. PMID:20347952

  4. Expression of Arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity.

    PubMed Central

    Hirschi, K D

    1999-01-01

    Calcium (Ca(2)+) efflux from the cytosol modulates Ca(2+) concentrations in the cytosol, loads Ca(2+) into intracellular compartments, and supplies Ca(2+) to organelles to support biochemical functions. The Ca(2+)/H(+) antiporter CAX1 (for CALCIUM EXCHANGER 1) of Arabidopsis is thought to be a key mediator of these processes. To clarify the regulation of CAX1, we examined CAX1 RNA expression in response to various stimuli. CAX1 was highly expressed in response to exogenous Ca(2+). Transgenic tobacco plants expressing CAX1 displayed symptoms of Ca(2+) deficiencies, including hypersensitivity to ion imbalances, such as increased magnesium and potassium concentrations, and to cold shock, but increasing the Ca(2+) in the media abrogated these sensitivities. Tobacco plants expressing CAX1 also demonstrated increased Ca(2+) accumulation and altered activity of the tonoplast-enriched Ca(2+)/H(+) antiporter. These results emphasize that regulated expression of Ca(2+)/H(+) antiport activity is critical for normal growth and adaptation to certain stresses. PMID:10559438

  5. Increasing marketability and profitability of product line thru PATRAN and NASTRAN

    NASA Technical Reports Server (NTRS)

    Hyatt, Art

    1989-01-01

    Starting with the design objective the operational cycle life of the Swaging Tool was increased. To accomplish this increase in cycle life without increasing the size or weight of the tool would be engineering achievement. However, not only was the operational cycle life increased between 2 to 10 times but simultaneously the size and weight of the Swage Tool was decreased by about 50 percent. This accomplishment now becomes an outstanding engineering achievement. This achievement was only possible because of the computerized Patran, Nastran and Medusa programs.

  6. Increased expression of TRPC4 channels associated with erectile dysfunction in diabetes.

    PubMed

    Sung, H H; Choo, S H; Ko, M; Kang, S J; Chae, M R; Kam, S C; Han, D H; So, I; Lee, S W

    2014-07-01

    In recent reports, an association between altered TRPC channel function and the development of various diabetic complications has drawn the attention of many investigators. The aim of this study was to investigate the expression of TRPC4 channels of corpus smooth muscle (CSM) cells in diabetes, and to evaluate the association between erectile dysfunction (ED) and altered TRPC4 channel function. The expression of TRPC4 in the penile tissue of human, normal and diabetic rat was investigated using RT-PCR, western blotting and immunohistochemistry (IHC). In vivo gene transfer of dominant negative (DN) TRPC4 into the CSM of rat was conducted. In vivo pelvic nerve stimulation was performed to measure erectile function. Expression of TRPC1, TRPC3, TRPC4 and TRPC6 in human and rat CSM tissues was confirmed by RT-PCR, western blot and IHC. In the diabetic rat, the expression levels of mRNA and protein of the TRPC4, and TRPC6 were significantly increased compared to control rats (p < 0.05). The change in TRPC4 expression in the diabetic rats was higher than those of the other TRPC subunits (p < 0.05). The IHC showed that only TRPC4 expression had a higher intensity in the diabetes compared to normal rats (p < 0.05). Gene transfection with TRPC4(DN) into the diabetic rats restored erectile function to levels similar to that of normal controls. Gene expression of TRPC4(DN) in CSM tissue was confirmed by RT-PCR 2 weeks after transfection. This study demonstrated that TRPC4 channel expression increased in the penile CSM cells of diabetic rats. The down-regulation of TRPC4 with DN form restored erectile function in the diabetic rats. The alteration of TRPC4 channel is one of pathophysiology of ED and could be a target for drug development for ED.

  7. Myotonic Dystrophy: Increased expression of the normal allele in CDM infants muscle

    SciTech Connect

    Radvanyi, H.H.; Gourdon, G.; Junien, C. |

    1994-09-01

    Myotonic dystrophy (DM) is an autosomal dominant multisystemic disorder characterized by a highly variable clinical phenotype. The mutation has been identified as an unstable trinucleotide CTG repeat in the 3{prime} untranslated region of the myotonin-protein kinase (MT-PK) gene. Congenital myotonic dystrophy (CDM), which represents the most severe phenotype, is exclusively maternally inherited. Recent studies, analysis by Northern blots and RT-PCR provided apparently conflicting results on the mutated allele expression in samples from congenitally affected children. The level of expression of the mutant allele depends on the extent of the repeat in the adult form and is no longer expressed when over 800-1300 repeats, whether in adult forms or in CDM. Could this decrease account for the late onset forms? However, the differences between the two phenotypes cannot be explained by the same mechanism. Alternatively, these differences could be due to differences in expression of the normal allele. We analyzed by quantitative RT-PCR the expression of the MT-PK gene in muscle samples from four CDM infants and two aged-matched normal controls. In two of these, the mutant allele (3.3 and 8 kb) was undetectable on Northern blots. We observed an increased expression of the MT-PK gene (10- to 20-fold) in tissues of severely affected congenital patients which can be attributed to the normal allele. Since expression of the normal allele is either normal or slightly decreased in the adult form, the dramatic increase in the congenital form could reflect a disturbance in muscle differentiation. Expression studies of MT-PK at different stages of development and, especially after the 20th week, are therefore required.

  8. Increased FOXP3 expression in tumour-associated tissues of horses affected with equine sarcoid disease.

    PubMed

    Mählmann, K; Hamza, E; Marti, E; Dolf, G; Klukowska, J; Gerber, V; Koch, C

    2014-12-01

    Recent studies suggest that regulatory T cells (Tregs) are associated with disease severity and progression in papilloma virus induced neoplasia. Bovine papilloma virus (BPV) is recognised as the most important aetiological factor in equine sarcoid (ES) disease. The aim of this study was to compare expression levels of Treg markers and associated cytokines in tissue samples of ES-affected equids with skin samples of healthy control horses. Eleven ES-affected, and 12 healthy horses were included in the study. Expression levels of forkhead box protein 3 (FOXP3), interleukin 10 (IL10), interleukin 4 (IL4) and interferon gamma (IFNG) mRNA in lesional and tumour-distant samples from ES-affected horses, as well as in dermal samples of healthy control horses were measured using quantitative reverse transcription polymerase chain reaction (PCR). Expression levels were compared between lesional and tumour-distant as well as between tumour-distant and control samples. Furthermore, BPV-1 E5 DNA in samples of ES-affected horses was quantified using quantitative PCR, and possible associations of viral load, disease severity and gene expression levels were evaluated. Expression levels of FOXP3, IL10 and IFNG mRNA and BPV-1 E5 copy numbers were significantly increased in lesional compared to tumour-distant samples. There was no difference in FOXP3 and cytokine expression in tumour-distant samples from ES- compared with control horses. In tumour-distant samples viral load was positively correlated with IL10 expression and severity score. The increased expression of Treg markers in tumour-associated tissues of ES-affected equids indicates a local, Treg-induced immune suppression.

  9. Apoptosis induced by tumor necrosis factor-alpha in rat hepatocyte cell lines expressing hepatitis B virus.

    PubMed Central

    Guilhot, S.; Miller, T.; Cornman, G.; Isom, H. C.

    1996-01-01

    Three well differentiated SV40-immortalized rat hepatocyte cell lines, CWSV1, CWSV2, and CWSV14, and Hepatitis B Virus (HBV)-producing cell lines derived from them were examined for sensitivity to tumor necrosis factor (TNF)-alpha. CWSV1, CWSV2, and CWSV14 cells were co-transfected with a DNA construct containing a dimer of the HBV genome and the neo gene and selected in G418 to generate stable cell lines. Characterization of these cell lines indicated that they contain integrated HBV DNA, contain low molecular weight HBV DNA compatible with the presence of HBV replication intermediates, express HBV transcripts, and produce HBV proteins. The viability of CWSV1, CWSV2, and CWSV2 cells was not significantly altered when they were treated with TNF-alpha at concentrations as high as 20,000 U/ml. The HBV-expressing CWSV1 cell line, SV1di36, and the HBV-expressing CWSV14 cell line, SV14di208, were also not killed when treated with TNF-alpha. However, the HBV-expressing CWSV2 cell line, SV2di366, was extensively killed when treated with TNF-alpha at concentrations ranging from 200 to 20,000 U/ml. Analysis of several different HBV-producing CWSV2 cell lines indicated that TNF-alpha killing depended upon the level of HBV expression. The TNF-alpha-induced cell killing in high HBV-producing CWSV2 cell lines was accompanied by the presence of an oligonucleosomal DNA ladder characteristic of apoptosis. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 9 Figure 10 Figure 11 PMID:8774135

  10. Increase in the scattering of electric field lines in a new high voltage SOI MESFET

    NASA Astrophysics Data System (ADS)

    Anvarifard, Mohammad K.

    2016-09-01

    This paper illustrates a new efficient technique to enhance the critical features of a silicon-on-insulator metal-semiconductor field-effect transistor (SOI MESFET) applied in high voltage applications. The structure we proposed utilizes a new method to scatter the electric field lines along the channel region. Realization of two trenches with different materials, which a trench is created in the channel region and the other one is created in the buried oxide, helps the proposed structure to improve the breakdown voltage, driving current, drain-source conductance, minimum noise figure, unilateral power gain and output power density. Exploring the obtained results, the proposed structure has superior electrical performance in comparison to the conventional structure.

  11. A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

    PubMed Central

    Pasumarthi, Ravi K.; Baridon, Brian; Djan, Esi; Trainor, Amanda; Griffey, Stephen M.; Engelhard, Eric K.; Rapp, Jared; Li, Bowen; de Jong, Pieter J.; Lloyd, K.C. Kent

    2015-01-01

    Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼80% of mutants showed specific staining in one or more tissues, while ∼20% showed no specific staining, ∼13% had staining in only one tissue, and ∼25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known. PMID:25591789

  12. Behavioral stress reduces RIP140 expression in astrocyte and increases brain lipid accumulation.

    PubMed

    Feng, Xudong; Lin, Yu-Lung; Wei, Li-Na

    2015-05-01

    Receptor-interacting protein 140 (RIP140) is highly expressed in the brain, and acts in neurons and microglia to affect emotional responses. The present study reveals an additional function of RIP140 in the brain, which is to regulate brain lipid homeostasis via its action in astrocytes. We found forced swim stress (FSS) significantly reduces the expression level of RIP140 and elevates cholesterol content in the brain. Mechanistically, FSS elevates endoplasmic reticulum stress, which suppresses RIP140 expression by increasing microRNA 33 (miR33) that targets RIP140 mRNA's 3'-untranslated region. Consequentially, cholesterol biosynthesis and export are dramatically increased in astrocyte, the major source of brain cholesterol. These results demonstrate that RIP140 plays an important role in maintaining brain cholesterol homeostasis through, partially, regulating cholesterol metabolism in, and mobilization from, astrocyte. Altering RIP140 levels can disrupt brain cholesterol homeostasis, which may contribute to behavioral stress-induced neurological disorders. PMID:25697398

  13. Behavioral stress reduces RIP140 expression in astrocyte and increases brain lipid accumulation.

    PubMed

    Feng, Xudong; Lin, Yu-Lung; Wei, Li-Na

    2015-05-01

    Receptor-interacting protein 140 (RIP140) is highly expressed in the brain, and acts in neurons and microglia to affect emotional responses. The present study reveals an additional function of RIP140 in the brain, which is to regulate brain lipid homeostasis via its action in astrocytes. We found forced swim stress (FSS) significantly reduces the expression level of RIP140 and elevates cholesterol content in the brain. Mechanistically, FSS elevates endoplasmic reticulum stress, which suppresses RIP140 expression by increasing microRNA 33 (miR33) that targets RIP140 mRNA's 3'-untranslated region. Consequentially, cholesterol biosynthesis and export are dramatically increased in astrocyte, the major source of brain cholesterol. These results demonstrate that RIP140 plays an important role in maintaining brain cholesterol homeostasis through, partially, regulating cholesterol metabolism in, and mobilization from, astrocyte. Altering RIP140 levels can disrupt brain cholesterol homeostasis, which may contribute to behavioral stress-induced neurological disorders.

  14. Cholesteryl ester hydroperoxides increase macrophage CD36 gene expression via PPAR{alpha}

    SciTech Connect

    Jedidi, Iness; Couturier, Martine; Therond, Patrice; Gardes-Albert, Monique; Legrand, Alain; Barouki, Robert; Bonnefont-Rousselot, Dominique; Aggerbeck, Martine . E-mail: Martine.Aggerbeck@univ-paris5.fr

    2006-12-22

    The uptake of oxidized LDL by macrophages is a key event in the development of atherosclerosis. The scavenger receptor CD36 is one major receptor that internalizes oxidized LDL. In differentiated human macrophages, we compared the regulation of CD36 expression by copper-oxidized LDL or their products. Only oxidized derivatives of cholesteryl ester (CEOOH) increased the amount of CD36 mRNA (2.5-fold). Both oxidized LDL and CEOOH treatment increased two to fourfold the transcription of promoters containing peroxisome-proliferator-activated-receptor responsive elements (PPRE) in the presence of PPAR{alpha} or {gamma}. Electrophoretic-mobility-shift-assays with nuclear extracts prepared from macrophages treated by either oxidized LDL or CEOOH showed increased binding of PPAR{alpha} to the CD36 gene promoter PPRE. In conclusion, CEOOH present in oxidized LDL increase CD36 gene expression in a pathway involving PPAR{alpha}.

  15. A Point Mutation in DNA Polymerase β (POLB) Gene Is Associated with Increased Progesterone Receptor (PR) Expression and Intraperitoneal Metastasis in Gastric Cancer

    PubMed Central

    Tan, Xiaohui; Wu, Xiaoling; Ren, Shuyang; Wang, Hongyi; Li, Zhongwu; Alshenawy, Weaam; Li, Wenmei; Cui, Jiantao; Luo, Guangbin; Siegel, Robert S.; Fu, Sidney W.; Lu, Youyong

    2016-01-01

    Increased expression of progesterone receptor (PR) has been reported in gastric cancer (GC). We have previously identified a functional T889C point mutation in DNA polymerase beta (POLB), a DNA repair gene in GC. To provide a detailed analysis of molecular changes associated with the mutation, human cDNA microarrays focusing on 18 signal transduction pathways were used to analyze differential gene expression profiles between GC tissues with T889C mutant in POLB gene and those with wild type. Among the differentially expressed genes, notably, PR was one of the significantly up-regulated genes in T889C mutant POLB tissues, which were subsequently confirmed in POLB gene transfected AGS cell line. Interestingly, patients with T889C mutation and PR positivity were associated with higher incidence of intraperitoneal metastasis (IM). In vitro studies indicate that PR expression was upregulated in AGS cell line when transfected with T889C mutant expression vector. Cotransfection of T889C mutant allele and PR gene induced cell migration in the cell line. These data demonstrated that T889C mutation-associated PR overexpression results in increased IM. Therefore, T889C mutation-associated PR overexpression may serve as a biomarker for an adverse prognosis for human GC. PMID:27471563

  16. Increased expression of estrogen-related receptor β during adaptation of adult cardiomyocytes to sustained hypoxia

    PubMed Central

    Cunningham, Kathryn F; Beeson, Gyda C; Beeson, Craig C; McDermott, Paul J

    2016-01-01

    Estrogen-related Receptors (ERR) are members of the steroid hormone receptor superfamily of transcription factors that regulate expression of genes required for energy metabolism including mitochondrial biogenesis, fatty acid oxidation and oxidative phosphorylation. While ERRα and EPPγ isoforms are known to share a wide array of target genes in the adult myocardium, the function of ERRβ has not been characterized in cardiomyocytes. The purpose of this study was to determine the role of ERRβ in regulating energy metabolism in adult cardiomyocytes in primary culture. Adult feline cardiomyocytes were electrically stimulated to contract in either hypoxia (0.5% O2) or normoxia (21% O2). As compared to baseline values measured in normoxia, ERRβ mRNA levels increased significantly after 8 hours of hypoxia and remained elevated over 24 h. Conversely, ERRβ mRNA decreased to normoxic levels after 4 hours of reoxygenation. Hypoxia increased expression of the α and β isoforms of Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1) mRNA by 6-fold and 3-fold, respectively. Knockdown of ERRβ expression via adenoviral-mediated delivery of ERRβ shRNA blocked hypoxia-induced increases in PGC-1β mRNA, but not PGC-1α mRNA. Loss of ERRβ had no effect on mtDNA content as measured after 24 h of hypoxia. To determine whether loss of ERRβ affected mitochondrial function, oxygen consumption rates (OCR) were measured in contracting versus quiescent cardiomyocytes in normoxia. OCR was significantly lower in contracting cardiomyocytes expressing ERRβ shRNA than scrambled shRNA controls. Maximal OCR also was reduced by ERRβ knockdown. In conclusion: 1) hypoxia increases in ERRβ mRNA expression in contracting cardiomyocytes; 2) ERRβ is required for induction of the PGC-1β isoform in response to hypoxia; 3) ERRβ expression is required to sustain OCR in normoxic conditions. PMID:27335690

  17. Tumor Necrosis Factor B (TNFB) Genetic Variants and Its Increased Expression Are Associated with Vitiligo Susceptibility

    PubMed Central

    Laddha, Naresh C.; Dwivedi, Mitesh; Gani, Amina R.; Mansuri, Mohmmad Shoab; Begum, Rasheedunnisa

    2013-01-01

    Genetic polymorphisms in TNFB are involved in the regulation of its expression and are found to be associated with various autoimmune diseases. The aim of the present study was to determine whether TNFB +252A/G (rs909253) and exon 3 C/A (rs1041981) polymorphisms are associated with vitiligo susceptibility, and expression of TNFB and ICAM1 affects the disease onset and progression. We have earlier reported the role of TNFA in autoimmune pathogenesis of vitiligo, and we now show the involvement of TNFB in vitiligo pathogenesis. The two polymorphisms investigated in the TNFB were in strong linkage disequilibrium and significantly associated with vitiligo. TNFB and ICAM1 transcripts were significantly increased in patients compared to controls. Active vitiligo patients showed significant increase in TNFB transcripts compared to stable vitiligo. The genotype-phenotype analysis revealed that TNFB expression levels were higher in patients with GG and AA genotypes as compared to controls. Patients with the early age of onset and female patients showed higher TNFB and ICAM1 expression. Overall, our findings suggest that the increased TNFB transcript levels in vitiligo patients could result, at least in part, from variations at the genetic level which in turn leads to increased ICAM1 expression. For the first time, we show that TNFB +252A/G and exon 3 C/A polymorphisms are associated with vitiligo susceptibility and influence the TNFB and ICAM1 expression. Moreover, the study also emphasizes influence of TNFB and ICAM1 on the disease progression, onset and gender bias for developing vitiligo. PMID:24312346

  18. Increased Cx32 expression in spinal cord TrkB oligodendrocytes following peripheral axon injury.

    PubMed

    Coulibaly, Aminata P; Isaacson, Lori G

    2016-08-01

    Following injury to motor axons in the periphery, retrograde influences from the injury site lead to glial cell plasticity in the vicinity of the injured neurons. Following the transection of peripherally located preganglionic axons of the cervical sympathetic trunk (CST), a population of oligodendrocyte (OL) lineage cells expressing full length TrkB, the cognate receptor for brain derived neurotrophic factor (BDNF), is significantly increased in number in the spinal cord. Such robust plasticity in OL lineage cells in the spinal cord following peripheral axon transection led to the hypothesis that the gap junction communication protein connexin 32 (Cx32), which is specific to OL lineage cells, was influenced by the injury. Following CST transection, Cx32 expression in the spinal cord intermediolateral cell column (IML), the location of the parent cell bodies, was significantly increased. The increased Cx32 expression was localized specifically to TrkB OLs in the IML, rather than other cell types in the OL cell lineage, with the population of Cx32/TrkB cells increased by 59%. Cx32 expression in association with OPCs was significantly decreased at one week following the injury. The results of this study provide evidence that peripheral axon injury can differentially affect the gap junction protein expression in OL lineage cells in the adult rat spinal cord. We conclude that the retrograde influences originating from the peripheral injury site elicit dramatic changes in the CNS expression of Cx32, which in turn may mediate the plasticity of OL lineage cells observed in the spinal cord following peripheral axon injury.

  19. Increased Cx32 expression in spinal cord TrkB oligodendrocytes following peripheral axon injury.

    PubMed

    Coulibaly, Aminata P; Isaacson, Lori G

    2016-08-01

    Following injury to motor axons in the periphery, retrograde influences from the injury site lead to glial cell plasticity in the vicinity of the injured neurons. Following the transection of peripherally located preganglionic axons of the cervical sympathetic trunk (CST), a population of oligodendrocyte (OL) lineage cells expressing full length TrkB, the cognate receptor for brain derived neurotrophic factor (BDNF), is significantly increased in number in the spinal cord. Such robust plasticity in OL lineage cells in the spinal cord following peripheral axon transection led to the hypothesis that the gap junction communication protein connexin 32 (Cx32), which is specific to OL lineage cells, was influenced by the injury. Following CST transection, Cx32 expression in the spinal cord intermediolateral cell column (IML), the location of the parent cell bodies, was significantly increased. The increased Cx32 expression was localized specifically to TrkB OLs in the IML, rather than other cell types in the OL cell lineage, with the population of Cx32/TrkB cells increased by 59%. Cx32 expression in association with OPCs was significantly decreased at one week following the injury. The results of this study provide evidence that peripheral axon injury can differentially affect the gap junction protein expression in OL lineage cells in the adult rat spinal cord. We conclude that the retrograde influences originating from the peripheral injury site elicit dramatic changes in the CNS expression of Cx32, which in turn may mediate the plasticity of OL lineage cells observed in the spinal cord following peripheral axon injury. PMID:27246301